smc5/6 complex subunit nse4a is involved in dna damage ... · 144 and nse4b promoters revealed that...

1

RESEARCH ARTICLE 1 2

The SMC5/6 Complex Subunit NSE4A is Involved in DNA Damage 3 Repair and Seed Development 4

5 Mariana Díaza,b,c, Petra Pecinkovaa, Anna Nowickaa,b,d, Célia Barouxe, Takuya 6 Sakamotoc, Priscilla Yuliani Gandhaa, Hana Jeřábkováb, Sachihiro Matsunagac, 7 Ueli Grossniklause & Ales Pecinkaa,b,1 8 aMax Planck Institute for Plant Breeding Research (MPIPZ), Cologne, Germany 9 bThe Czech Academy of Sciences, Institute of Experimental Botany (IEB), Centre of the 10 Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech 11 Republic 12 cDepartment of Applied Biological Science, Faculty of Science and Technology, Tokyo 13 University of Science, Chiba, Japan 14 dPolish Academy of Sciences, Franciszek Gorski Institute of Plant Physiology, Krakow, 15 Poland 16 eDepartment of Plant and Microbial Biology and Zürich-Basel Plant Science Center, 17 University of Zürich, Zürich, Switzerland18 1Address correspondence to [email protected] 19

20 Short title: Analysis of NSE4 genes in Arabidopsis 21

22 One sentence summary: Characterization of the two NSE4 paralogs in Arabidopsis 23 revealed that NSE4A is essential for both genome stability and plant reproduction. 24

25 The author responsible for distribution of materials integral to the findings presented in 26 this article in accordance with the policy described in the Instructions for Authors 27 (www.plantcell.org) is: Ales Pecinka ([email protected]). 28

29 ABSTRACT 30 The maintenance of genome integrity over cell divisions is critical for plant 31 development and the correct transmission of genetic information to the progeny. A key 32 factor involved in this process is the STRUCTURAL MAINTENANCE OF 33 CHROMOSOME 5/6 (SMC5/6) complex, related to the cohesin and condensin 34 complexes that control sister chromatid alignment and chromosome condensation, 35 respectively. Here we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the 36 SMC5/6 complex in Arabidopsis thaliana. NSE4A is expressed in meristems and 37 accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are 38 viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a 39 mutants produce abnormal seeds, with non-cellularized endosperm and embryos that 40 maximally develop to the heart or torpedo stage. This phenotype resembles the defects 41 in cohesin and condensin mutants and suggests a role for all three SMC complexes in 42 differentiation during seed development. By contrast, NSE4B is expressed in only a few 43 cell types and loss-of-function mutants do not have any obvious abnormal phenotype. In 44 summary, our study shows that the NSE4A subunit of the SMC5/6 complex is essential 45 for DNA damage repair in somatic tissues and plays a role in plant reproduction. 46

47

48

INTRODUCTION 49

Plant Cell Advance Publication. Published on April 29, 2019, doi:10.1105/tpc.18.00043

©2019 American Society of Plant Biologists. All Rights Reserved

2

The eukaryotic nuclear genome is packaged into higher-order chromatin structures, 50

which are dynamically remodeled during cellular activities (Alabert and Groth, 2012). 51

Key factors establishing and orchestrating chromosome organization are 52

STRUCTURAL MAINTENANCE OF CHROMOSOME (SMC) complexes - cohesin 53

(containing SMC1 and SMC3), condensin (containing SMC2 and SMC4), and the 54

SMC5/6 complex (containing SMC5 and SMC6) (reviewed in Hirano 2006; Jeppsson, 55

Kanno, et al. 2014; Uhlmann 2016). The heterodimeric SMC backbone serves as a 56

structural component and a docking platform for additional subunits that vary 57

depending on the complex, thereby enabling a variety of specific assemblies (reviewed 58

in Kegel and Sjögren 2010; Diaz and Pecinka 2018). Studies in yeasts and animals 59

showed that cohesin facilitates sister chromatin cohesion, and condensin I and II 60

complexes mediate large-scale chromatin folding and chromosome condensation 61

(reviewed in Hirano, 2012; Uhlmann, 2016). The major activity of the SMC5/6 complex 62

is the maintenance of nuclear genome stability by resolving complex structures and 63

possibly acting as antagonist of the cohesin complex (reviewed in de Piccoli et al., 64

2009; Kegel and Sjögren, 2010; Diaz and Pecinka, 2018). The SMC5/6 complex 65

performs many functions, such as the control of unidirectional rDNA replication, 66

neutralizing toxic DNA intermediates during replication, preventing homologous 67

recombination between non-homologous sequences, and alternative telomere 68

lengthening (Potts and Yu, 2007; Torres-Rosell et al., 2007; Chiolo et al., 2011; Menolfi 69

et al., 2015). 70

The SMC5/6 complex can be associated with up to six NON-STRUCTURAL 71

ELEMENT (NSE) subunits, which assemble in a combinatorial manner to form three 72

sub-complexes (NSE1-NSE3-NSE4, NSE5-NSE6, and NSE2-SMC5-SMC6) in yeasts 73

(de Piccoli et al., 2009; Duan et al., 2009). Studies in budding yeast, fission yeast and 74

mammalian cell cultures revealed that the NSE1-NSE3-NSE4 sub-complex binds 75

double stranded DNA and acts as a binding platform for the heads of SMC5 and SMC6 76

(Hudson et al., 2011; Palecek and Gruber, 2015; Zabrady et al., 2015). The least 77

evolutionary conserved SMC5/6 subunits are NSE5 and NSE6. They interact with the 78

SMC5-SMC6 hinges in budding yeast but with their heads in fission yeast (Pebernard et 79

al., 2006; de Piccoli et al., 2009; Duan et al., 2009). Recently, functional orthologs of 80

NSE5 and NSE6 have been identified in plants and mammals (Yan et al., 2013; Räschle 81

et al., 2015), but their molecular functions remain unclear. The NSE2-containing sub-82

complex has very important enzymatic activity: NSE2 (aka METHANE 83

3

METHYLSULFONATE SENSITIVE21, MMS21; aka HIGH PLOIDY2, HPY2) is 84

anchored to SMC5 and has SMALL UBIQUITIN-RELATED MODIFIER (SUMO) E3 85

ligase activity (Zhao and Blobel, 2005). Many proteins were found to be targets of 86

NSE2 SUMOylation, including several SMC5/6 and cohesin subunits, as well as DNA 87

repair proteins in plants, fungi, and animals (Zhao and Blobel, 2005; Potts and Yu, 88

2007; Pebernard et al., 2006; Ishida et al., 2009; Huang et al., 2009). 89

Homologs of all SMC5/6 subunits were identified in Arabidopsis thaliana 90

(Arabidopsis) (Schubert, 2009; Watanabe et al., 2009; Yan et al., 2013; Diaz and 91

Pecinka, 2018). However, our understanding of biological processes controlled by the 92

individual SMC5/6 subunits remains limited in plants. Arabidopsis plants mutated in 93

SMC6B (aka MIM - HYPERSENSITIVE TO MMS, IRRADIATION AND MMC) are 94

indistinguishable from the wild type under ambient conditions, but are hypersensitive to 95

DNA damaging treatments, show a delayed repair of DNA strand breaks, and have a 96

reduced frequency of homologous recombination (Mengiste et al., 1999; Kozak et al., 97

2009; Watanabe et al., 2009; Liu et al., 2015). smc6a mutants are viable even under 98

severe DNA damage but smc6a smc6b double mutation is embryo lethal (Watanabe et 99

al., 2009; Yan et al., 2013), indicating partial functional redundancy. Plants defective in 100

NSE2 are hypersensitive to DNA damage and display a wide range of pleiotropic 101

phenotypes, including leaf and stem malformations, branching defects, reduced 102

meristem size, impaired development of gametes, shortened vegetative phase, and 103

increased drought tolerance (Yuan et al., 2014; Huang et al., 2009; Ishida et al., 2009; 104

Xu et al., 2013; Zhang et al., 2013; Liu et al., 2014; Kwak et al., 2016). SMC5, SMC6, 105

and NSE1-4 are evolutionary conserved proteins. In addition, there are two other 106

SMC5/6 complex subunits (collectively named as NSE5 and NSE6) in fungi, humans, 107

and plants, which are presumably functionally conserved, but share little sequence 108

similarity (reviewed in Diaz and Pecinka 2018). In Arabidopsis, both the regulator of 109

systemic acquired resistance SUPPRESSOR OF NPR1-1, INDUCIBLE1 (SNI1) and 110

the ARABIDOPSIS SNI1 ASSOCIATED PROTEIN1 (ASAP1) were found in a 111

complex with SMC5 and SMC6B, and were thus proposed as the putative functional 112

orthologs of yeast NSE6 and NSE5, respectively (Yan et al., 2013). Both genes 113

participate in the control of genome stability and suppression of immune hyper-114

responses, which is a novel and unexpected function of the complex. 115

The variety of plant phenotypes seen in mutants affecting the SMC5/6 complex 116

suggests that it participates in multiple developmental and cellular pathways possibly 117

4

linked to stress responses. Currently, the composition of the plant SMC5/6 complex, the 118

roles of individual subunits, and their functional requirement in cellular and 119

developmental processes (beside DNA damage repair) are poorly characterized. In an 120

effort to obtain a more comprehensive functional understanding of the Arabidopsis 121

SMC5/6 complex, we characterized the roles of the NSE4A and NSE4B subunits. We 122

show that NSE4A is involved in repair of zebularine-induced DNA damage in 123

challenged somatic tissues. In addition, NSE4A is essential for reproductive 124

development in Arabidopsis, while the function of NSE4B remains elusive. 125

126

127

128

5

RESULTS 129

The NSE4 gene is duplicated in the Arabidopsis genome 130

The Arabidopsis genome contains two uncharacterized, putative NSE4 homologs: 131

NSE4A (At1g51130 encoding a 403 amino acid protein) and NSE4B (At3g20760 132

encoding a 383 amino acid protein) sharing 65.1% identity at the amino acid level 133

(Figure 1A,B). To identify the age of this duplication, we built a NSE4 phylogeny 134

across green plants using the Schizosaccharomyces pombe and Homo sapiens NSE4s as 135

outgroups (Figure 1C; Supplemental Table 1; Supplemental Data Sets 1-3). Except for 136

mosses and liverworts, which carry a single NSE4, all other plant genomes contained at 137

least two NSE4 copies. Orthologs of A. thaliana NSE4A and NSE4B occurred in A. 138

lyrata, Capsella rubella, and Eutrema salsugineum. The only exception was Brassica 139

rapa, where both NSE4 copies of were derived from NSE4A, while NSE4B was missing. 140

This suggests that the NSE4A and NSE4B originate from the whole genome duplication 141

event which occurred ~47 million years ago (MYA) and preceded radiation of the 142

species within Brassicaceae (Kagale et al., 2014). Phylogenetic shadowing of NSE4A 143

and NSE4B promoters revealed that both contain conserved blocks, A1 and B1, 144

respectively, directly upstream of the transcription start site (Supplemental Figure 1; 145

Supplemental Table 2). However, the A1 block was clearly larger and more similar 146

between species, indicating that it may contain key NSE4A cis-regulatory sequences. 147

There was another set of conserved NSE4 paralogs in Poaceae, including 148

Brachypodium distachyon, Hordeum vulgare, and Zea mays (Figure 1C). These 149

paralogs most likely appeared during the Poaceae-specific whole genome duplication 150

event ~70 MYA (Paterson et al., 2009). We found a total of six NSE4 copies in rice and 151

four in tomato. Some of these copies were short and grouped with more distantly related 152

species (Figure 1C), raising questions on their origin and functionality. The high 153

frequency of multiple NSE4 copies per genome may indicate rapid NSE4 sub- or neo-154

functionalization in different plant lineages. 155

To assess the role of the NSE4 genes in plant growth and development, we 156

isolated T-DNA insertion mutations in NSE4A and NSE4B (Figure 1A). The nse4a-1 157

allele carried a T-DNA in the second exon and was lethal as indicated by the absence of 158

homozygous mutants in the progeny of heterozygous parents. However, we recovered 159

viable homozygous nse4a-2 plants carrying a T-DNA insertion in the last exon, 56 160

amino acids before the stop codon (Supplemental Figure 2). 3' RACE revealed that the 161

NSE4A transcript in nse4a-2 plants continued into the T-DNA and maintained the 162

6

reading frame for 201 nucleotides, adding a predicted 67 alien amino acids to the 163

7

NSE4A protein produced by nse4a-2 mutants. Therefore, nse4a-2 most likely represents 164

a partial loss-of-function allele with a modified C-terminus. Juvenile and non-flowering 165

nse4a-2 plants were smaller than the wild type (Figures 1D and 1E) but recovered and 166

were indistinguishable from control plants at flowering (Figure 1F). The nse4b mutant 167

alleles carried T-DNA insertions in the second intron (nse4b-1) and the fifth exon 168

(nse4b-2), respectively. Amplification from cDNA with primer pairs positioned on 169

either side of the T-DNA insertions yielded very low or no products in quantitative PCR, 170

suggesting that both insertions disrupt the NSE4B transcript (Supplemental Figure 3). 171

However, both nse4b-1 and nse4b-2 plants were viable and resembled wild-type plants 172

(Figures 1D to 1F). Combining the nse4a-2 and nse4b-2 alleles in a homozygous double 173

mutant resulted in a nse4a-2-like phenotype, suggesting that NSE4A and NSE4B do not 174

act redundantly during vegetative development. 175

To reveal the activity pattern of the NSE4 promoter, we generated stable reporter 176

lines where the NSE4A and NSE4B promoters were fused to the uidA gene encoding β-177

glucuronidase (GUS) (ProNSE4A:GUS and ProNSE4B:GUS). The NSE4A promoter 178

was strongly active in emerging true leaves and weakly active in the vasculature of the 179

cotyledons at seven days after germination (DAG; Figure 1G). In addition, we observed 180

signals in the stele tissues within the differentiation zone of the root, but there was no 181

ProNSE4A activity in root meristems. At 14 DAG, ProNSE4A was weakly active in all 182

aerial tissues (Figure 1H). Flowers showed ProNSE4A:GUS activity in sepals, the upper 183

half of fully elongated anther filaments, pistils, and anthers (Figure 1I, top row). By 184

contrast, ProNSE4B:GUS activity was restricted to the leaf stipules and a small domain 185

in the root apical meristem at 7 DAG (Figure 1G, red arrowheads and insets). This 186

pattern remained unchanged during the entire vegetative phase (Figure 1H). In flowers, 187

ProNSE4B was active in anthers between stages 10 and 12 (Figure I). The difference in 188

the expression patterns of NSE4A and NSE4B could be due to the association of the 189

endogenous NSE4B locus with repressive histone H3 lysine 27 tri-methylation. 190

(Supplemental Figures 4 and 5). 191

192

NSE4A is expressed in pollen, ovules, and seeds 193

The activity of ProNSE4A and ProNSE4B in flowers prompted us to analyze the 194

reproductive stages in more detail. To get better insight into the expression of the 195

NSE4A protein, we expressed a translational fusion of NSE4A with VENUS (an 196

improved variant of the yellow fluorescent protein; Nagai et al., 2002) under the control 197

8

of its native promoter (ProNSE4A:NSE4A-VENUS) in the nse4a-2 background. Based 198

on the full complementation of nse4a-2 hypersensitivity to zebularine (Figure 2A), we 199

conclude that the addition of VENUS does not interfere with NSE4A function. 200

Analysis of the transcription during pollen development revealed strong and weak 201

activity of ProNSE4A and ProNSE4B, respectively (Figures 2B and 2C). The 202

microspores (flower stage 10; Bowman 1994) showed on average the strongest signals 203

9

for both ProNSE4A:GUS and ProNSE4B:GUS, which decreased over subsequent 204

developmental stages. There was practically no transcriptional activity of both genes in 205

mature pollen from open anthers (flower stage 14). At the protein level, NSE4A was 206

present at all pollen stages in the cell lineage leading to the sperm cells, as indicated by 207

VENUS signals in the single nucleus of the unicellular microspore, the generative 208

nucleus of bicellular pollen (flower stage 11), and the two sperm nuclei of tricellular 209

pollen (Figure 2B). No NSE4A-VENUS signal could be observed in the vegetative 210

nucleus. 211

During ovule development (Figure 2D), we observed ProNSE4A:GUS activity in 212

ovule primordia at flower stage 10, the nucellus at stage 11, and the embryo sac in 213

stages 12 to 14. The transcriptional profile was largely in agreement with NSE4A 214

protein accumulation (Figure 2E). Strong NSE4A-VENUS signals were observed in 215

almost all cells of the nucellus except for the megaspore mother cell, where the fusion 216

protein was barely detectable (Figure 2E, flower stage 10, arrowhead). However, 217

NSE4A-VENUS accumulated strongly in female meiocytes initiating meiotic prophase 218

I (Figure 2E, flower stage 11, arrowhead). The differences between GUS and VENUS 219

signals could be due to different stability of GUS mRNA and/or protein as compared to 220

NSE4A-VENUS transcript and/or protein. After pollination, ProNSE4A activity was 221

detected in the embryo and the chalazal endosperm, and later (at 96 h after pollination) 222

also in the syncytial endosperm (Figure 2F). This corresponds well with the strong 223

NSE4A-VENUS signals in developing embryos (Figure 2G), and also the prominent 224

localization to the nuclei of the syncytial endosperm (Figure 2G). By contrast, 225

ProNSE4B activity during early ovule development remained largely below detection 226

limit (Figure 2D) and we detected weak activity only in mature embryo sacs, with GUS 227

activity getting stronger after pollination, leading to a clear signal in the early embryo 228

up to the globular stage (Figure 2F). 229

In summary, these results confirmed NSE4A to be a nuclear protein, as expected 230

for a DNA repair factor, and revealed a dynamic expression pattern of NSE4A during 231

sporogenesis, gametogenesis, embryogenesis, and endosperm development. The high 232

levels of NSE4A during meiosis and in the proliferating fertilization products may be 233

linked with its DNA repair function, e.g. during meiotic crossing-over or to ensure 234

genome integrity during the fast mitoses in embryo and endosperm. 235

236

NSE4A plays a role in seed development 237

10

Prompted by NSE4 expression in seeds, we analyzed fertility of nse4a and nse4b 238

mutants two weeks after pollination (Figures 3A and 3B). In contrast to wild-type 239

plants, siliques from nse4a-1/NSE4A heterozygotes produced 28.8% abnormal seeds 240

(pale seeds representing delayed embryos and/or aborted seeds; n = 1402, Figures 3A 241

and 3B). Fertility was even more impaired in homozygous nse4a-2 plants, with 242

approximately half (53.4%) of the seeds developing normally, 22% early aborted 243

ovules, and 24.6% abnormally large seeds with a glossy surface and liquid endosperm 244

(n = 1008). Clearing of abnormal nse4a-1 and nse4a-2 seeds revealed that the embryos 245

were arrested at the heart or heart-to-torpedo transition stages, respectively (Figure 3C; 246

Supplemental Figure 6A). A NSE4A genomic construct could fully rescue the nse4a-2 247

mutant seed phenotype (up to 96.5% normal seeds, n = 949), confirming that embryo 248

unviability is a consequence of the loss of NSE4A function (Figures 3A and 3B). To test 249

whether the increased frequency of abnormal seeds in nse4a-1 heterozygous plants 250

(28.8% observed versus expected 25%) is due to preferential transmission of the mutant 251

allele or a partial gametophytic maternal effect, nse4a-1/NSE4A heterozygous plants 252

were self-pollinated and reciprocally crossed to wild-type plants. The frequency of late 253

aborted seeds resulting from these crosses was scored (Supplemental Figure 6B). 254

Reciprocal crosses resulted in 0.6% to 2.0% of late aborted seeds, indistinguishable 255

from the wild-type control, while self-pollinated nse4a-1/NSE4A heterozygous plants 256

produced 23.9% late aborted seeds. These results indicate that nse4a-1 is a zygotic 257

embryo-lethal mutation. By contrast, and in agreement with the NSE4B expression 258

pattern, nse4b-1 and nse4b-2 single mutants were fully fertile, while the nse4a-2 nse4b-259

2 double mutant showed a similar phenotype as the nse4a-2 single mutant (Figures 3A 260

and 3B). Hence, NSE4A is required for normal seed development, while NSE4B is 261

dispensable. 262

263

NSE4A is involved in somatic DNA damage repair 264

Next, we tested which of the Arabidopsis NSE4 paralogs is involved in DNA damage 265

repair. First, we scored for the transcriptional response of NSE4A and NSE4B to drug 266

treatment using the promoter-GUS reporter lines (Figure 4). No induction was observed 267

for ProNSE4B:GUS upon treatment with DNA damaging agents including zebularine 268

(10 µM), which (similarly to the related drug 5-azacytidine; reviewed in Stingele and 269

Jentsch, 2015; Tretyakova et al., 2015) generates enzymatic DNA-protein crosslinks by 270

covalently trapping DNMT1 class enzymes, and bleocin (25 nM), which causes DNA 271

11

strand breaks (Figures 4A and 4C). By contrast, ProNSE4A became active throughout 272

the entire meristematic zone and in the emerging lateral roots (Figures 4A and 4C), 273

indicating that NSE4A is activated by different types of DNA damage. This 274

transcriptional activation was accompanied by protein accumulation as indicated by 275

NSE4A-VENUS signals within a larger area of the root apical meristem of stressed 276

reporter plants (Figure 4B). 277

Subsequently, we assessed the functional contribution of the NSE4 genes to plant 278

survival upon drug-induced DNA damage. To this aim, we monitored the growth of 279

wild-type, nse4a-2 single mutant, nse4a-2 complemented with NSE4A genomic 280

construct (ProNSE4A:NSE4A:TerNSE4A), nse4b (both alleles), and nse4a-2 nse4b-2 281

double mutant plants on media containing 10 µM zebularine, 50 nM bleocin, 10 µM 282

MMC, or 1 mM hydroxyurea (HU) (Figures 5A and 5B; Supplemental Figure 7). In a 283

separate assay, we applied the DNA alkylating agent methyl methane sulfonate (MMS) 284

(Figure 5C), which caused poor growth of the Arabidopsis smc6b-3 (mim-1) mutant 285

(Mengiste et al., 1999). As positive controls we used the drug-sensitive ATAXIA 286

12

TELANGIECTASIA-MUTATED AND RAD3-RELATED (ATR) signaling kinase mutant 287

(atr-2), the DNA LIGASE4 mutant (lig4-2), WEE1 KINASE HOMOLOG mutant (wee1-288

13

1), and mutants in the two SMC5/6 complex subunits, SMC6B (smc6b-1) and HPY2 289

(hpy2-2) (De Schutter et al., 2007; Ishida et al., 2009; Yuan et al., 2014; Liu et al., 290

14

2015). The nse4b-1 and nse4b-2 single mutants were not hypersensitive to any of the 291

applied genotoxic treatments (Figures 5A to 5C). The nse4a-2 single and nse4a-2 292

nse4b-2 double mutants were indistinguishable from the wild type under MMC, bleocin, 293

and HU stress, but were strongly hypersensitive to zebularine and MMS (Figures 5A to 294

5C). By contrast, smc6b-1 was also hypersensitive to MMC treatment, which could be 295

due to the fact that nse4a-2 is only a partial-loss-of-function allele. To test for effect on 296

homologous recombination (HR) rates, we generated nse4a-2 nse4b-2 double mutants 297

carrying the reporter N1DC1 no. 11 (B11) with 566 bp overlap of GUS recombination 298

substrate in direct orientation (Puchta et al., 1995). The plants were grown for 10 days 299

in media containing low amounts of zebularine (1.25 and 2.5 µM) to avoid lethality. We 300

used multiple independent lines of each analyzed genotype, which showed a zebularine 301

dose-dependent increase in HR rate, but no significant differences between wild-type, 302

nse4a-2, and nse4b-2 lines (Figure 5D). This result differs from those published for 303

hpy2 and smc6 mutants, which showed reduced HR rates (Mengiste et al., 1999; 304

Watanabe et al., 2009; Yuan et al., 2014). On the one hand, this may suggest that NSE4 305

proteins are not controlling single strand annealing type of HR in Arabidopsis. On the 306

other hand, these results should be interpreted with caution because nse4a-2 is not a null 307

allele and nse4b mutants are not sensitive to DNA damage treatments. 308

Inhibition of root growth in response to DNA damage is frequently accompanied 309

by increased cell death. Therefore, we monitored the amount of dead cells using the 310

propidium iodide (PI) uptake assay in control and 20 µM zebularine-treated plants 311

(Figure 5E). While there were no or few dead cells in wild-type and nse4b-2 plants, 312

nse4a-2 single and nse4a-2 nse4b-2 double mutant plants showed a drastic increase 313

upon zebularine treatment. The drug sensitivity phenotype (growth and cell death) of 314

nse4a-2 to zebularine is directly due to the loss of NSE4A activity as shown by 315

complementation using an NSE4A genomic construct (Figures 5A, 5B, and 5E). We 316

noticed that the root meristem was partially disorganized in zebularine-treated nse4a-2 317

plants. Therefore, we estimated the meristem size by counting the number of cells in the 318

cortex layer between the quiescent center and the differentiation zone (Figure 5F). 319

Wild-type and nse4b-2 roots contained 38 to 45 cells, and this number did not change 320

significantly after 24 h of 20 µM zebularine treatment (ANOVA, post hoc Duncan’s 321

test, P > 0.05). By contrast, nse4a-2 showed a significant 31% reduction to 26 cells 322

upon zebularine treatment. To test the effect of the mutation on cell cycle regulation, we 323

introduced a G2/M DNA damage reporter, which utilizes a translational fusion between 324

15

CyclinB1;1 and GUS (Colón-Carmona et al., 1999), into nse4a-2 and nse4b-2 mutant 325

backgrounds. The chimeric protein accumulates specifically in G2 phase of cycling cells 326

and is destroyed at the onset of mitosis resulting in a loss of the signal. Double 327

homozygous lines were exposed to 10 µM zebularine for up to 48 h, and the domain of 328

GUS expression was monitored (Figure 5G). The nse4a-2 roots showed an increased 329

number of GUS-positive cells already at 0 h, indicating a prolonged G2 phase. After 48 330

h treatment, meristems of nse4a-2 plants were damaged, as indicated by an abnormal 331

root morphology and root hairs emerging close to the root tips. The response in nse4b-2 332

and wild-type was slower, less severe, and similar between the two (Figure 5G). 333

Collectively, these results demonstrate that NSE4A responds to genotoxic stress, is 334

likely involved in DNA repair of zebularine-induced DNA-protein crosslinks, and is 335

required to promote cell division in response to this genotoxic drug, possibly to actively 336

propagate cells after repair. 337

338

Loss of NSE4A function causes up-regulation of DNA damage repair and immune 339

response genes 340

We analyzed effect of the nse4a-2 mutation on gene expression by RNA-sequencing 341

using dissected shoot apices from 10-day-old wild-type and nse4a-2 plants treated as 342

without (mock) and with 20 μM zebularine for 24 h (Figure 6; Supplemental Data Set 343

4). In mock-treated nse4a-2 we identified 555 significantly up- and 181 significantly 344

down-regulated genes relative to mock-treated wild type (Figure 6A; DESeq; adjusted P 345

< 0.05; the same parameters apply to the whole section). In zebularine- relative to 346

mock-treated wild-type plants we found 446 significantly up- and 183 significantly 347

down-regulated genes, i.e. many more than we identified in a previous study (Liu et al., 348

2015). This difference is most likely due to the treatment in liquid media, allowing for a 349

more intense uptake of zebularine in comparison to the previously used solid media. 350

Zebularine treatment of nse4a-2 plants had the strongest effect, leading to up-regulation 351

of 1374 and down-regulation of 773 genes in comparison to mock-treated nse4a-2 352

control plants. Up-regulated genes included several prominent DNA damage repair 353

markers (Figure 6B). These data suggest that the SMC5/6 complex is not required for 354

transcriptional up-regulation of DNA damage repair genes, but loss of its functionality 355

triggers a more intense DNA damage response (Figure 6B). 356

Previous microarray-based expression analysis of sni1-1 suggested a link between 357

function of the SMC5/6 complex and immune responses (Mosher et al., 2006). 358

16

Comparison of the transcriptomes from nse4a-2 and sni1-1 mutants revealed 82 (5.8%) 359

commonly up- and 6 (0.5%) commonly down-regulated genes (Figure 6C; 360

Supplemental Data Set 5). The up-regulated genes were mainly associated with stress 361

responses, defense responses to (biotic) stimuli, and responses to other organisms 362

(Figure 6D; Supplemental Table 3), which was described for SNI1 (Mosher et al., 2006; 363

Yan et al., 2013), but is new information for NSE4A. The up-regulated genes in nse4a-2 364

plants included PATHOGENESIS-RELATED GENE 2 (PR2 aka BETA-1,3-365

17

GLUCANASE2), PR4, PR5 and several TIR-NBS-LRR genes (At5g46490, WRR4, 366

At3g44630; Figure 6E; Supplemental Data Set 5). This indicates that mutations 367

affecting the SMC5/6 complex cause constitutive expression of immune response genes 368

and lead to activation of other DNA damage repair pathways, most likely due to 369

accumulation of spontaneous DNA damage. 370

371

NSE4A and NSE4B interact with the same SMC5/6 complex subunits 372

In plants, the architecture of SMC5/6 complex remains unknown. Based on fungal and 373

animal models, we assume that NSE4 may act as a central subunit interacting with 374

SMC5 and SMC6, and possibly several other NSEs (Duan et al., 2009; Hudson et al., 375

2011). To test whether this hypothesis holds true for both NSE4 paralogs, we performed 376

yeast-two-hybrid (Y2H) assays. The assay conditions were optimized using the positive 377

(T+53) and the negative (T+lam C) controls, and we suppressed protein auto-activation 378

by adjusting the 3-Amino-1,2,4-triazole (3-AT) concentrations (Figure 7A; 379

Supplemental Figure 8; Supplemental Table 4). As a control, we confirmed the 380

interaction of SMC6A and SMC6B hinges with the SMC5 hinge (Figure 7A). 381

Subsequently, we tested for interactions of full-length SMC5 or SMC6 with NSE4A and 382

NSE4B. While the interaction between both NSE4 paralogs and SMC5 was positive 383

(Figure 7A), we did not observe yeast growth when testing interactions with SMC6A 384

and SMC6B. This remained true even after switching the tag positions (N- and C-385

terminal) and extensive optimization (Supplemental Figure 8). Within the NSE1-NSE3-386

NSE4 subcomplex, we measured positive interactions of both NSE4 paralogs with 387

NSE3, and confirmed (after Li et al. 2017) the interaction of NSE1 with NSE3 (Figure 388

7A). However, we did not detect interactions between NSE4A or NSE4B and NSE1. To 389

validate the interactions identified by Y2H, we performed Bimolecular Fluorescence 390

Complementation (BiFC) assays in Nicotiana benthamiana and analyzed signals using 391

confocal microscopy (Figure 7B). In all cases, the signals were localized to the nucleus 392

and confirmed that both NSE4A and NSE4B are able to interact with SMC5 and NSE3. 393

Moreover, we tested protein-protein interactions using co-immunoprecipitation (Co-IP) 394

assays in N. benthamiana and validated (i) the interactions of the SMC5 hinge with the 395

hinges of SMC6A and SMC6B; (ii) the interaction of NSE3 with NSE4A and NSE4B; 396

and (iii) the interaction of NSE3 with NSE1 (Figure 7C, left panel; Supplemental Figure 397

9). We could not evaluate the interactions of NSE4A and NSE4B with the full-length 398

SMC5 protein using Co-IPs because, despite extensive optimization, SMC5 did not 399

18

reach detectable levels following transfection in N. benthamiana leaves as assayed by 400

Western blotting. However, the presence of tagRFP-SMC5 modified the nuclear 401

distribution of both NSE4A-EYFP and NSE4B-EYFP from a dispersed to a speckled 402

pattern (Figure 7C, right panel). 403

19

In summary, the results from Y2H, BiFC, and Co-IP assays together with 404

published data allow us to conclude that individual Arabidopsis SMC5/6 subunits 405

interact and that SMC5 recruits NSE4A and NSE4B into speckled domains in the 406

nucleus (Figure 7C). Based on these experiments, we developed a model for 407

interactions between SMC5/6 subunits in Arabidopsis (Figure 7D). 408

409

The NSE4B protein can partially substitute NSE4A protein functions 410

The NSE4A and NSE4B paralogs show little overlap in their expression patterns and 411

loss-of-function phenotypes. To test whether NSE4A and NSE4B also diverged 412

functionally, we developed a promoter swap construct consisting of the NSE4B genomic 413

coding sequence under the control of the NSE4A promoter 414

(ProNSE4A:GenomicNSE4B:TerNSE4B). This construct was transformed into 415

homozygous nse4a-2 plants and individuals heterozygous or homozygous for the 416

promoter swap construct were selected in the T2 generation and tested for zebularine 417

sensitivity in the T3 generation. While the control nse4a-2 plants were strongly 418

hypersensitive, several independent promoter swap lines showed rescue, albeit 419

incomplete, of the drug sensitivity phenotype with average roots length being 420

intermediate between those of nse4a-2 and wild-type plants (Figures 8A and 8B). 421

In addition, the broader expression domain of NSE4B in the promoter swap lines 422

was able to rescue the seed abortion phenotype of nse4a-2 (Figures 8C and 8D). 423

Furthermore, NSE4B expression in the nse4a-1 background allowed the recovery of 424

homozygous nse4a-1 plants (24% viable nse4a-1/nse4a-1 plants in the progeny of a 425

NSE4A/nse4a-1; ProNSE4A:GenomicNSE4B:TerNSE4B segregating parent, n=92, 426

Supplemental Tables 5 and 6). 427

Taken together, these results demonstrate that NSE4A and NSE4B have similar 428

biochemical activities, which are fully exchangeable during seed development but only 429

partially in DNA damage responses. 430

431

432

433

20

DISCUSSION 434

The SMC5/6 complex plays a crucial role in the maintenance of genome stability in 435

eukaryotes (de Piccoli et al., 2009; Kegel and Sjögren, 2010; Jeppsson et al., 2014b; 436

Diaz and Pecinka, 2018) . Some of its subunits remain poorly characterized in plants, 437

including the two NSE4 homologs. Here, we demonstrate that NSE4A is involved in 438

preserving genome stability and controls seed development. NSE4B is barely active 439

during normal development and non-responsive to drug-induced genotoxic stress. 440

441

NSE4A is an essential gene in Arabidopsis 442

The NSE4 paralogs of A. thaliana originate from the whole-genome duplication event 443

(alpha) that occurred ~47 MYA in Brassicaceae (Kagale et al., 2014). Surprisingly, 444

there were at least two NSE4A copies in all vascular plants analyzed, with the highest 445

number of six copies in rice. The NSE4 amplifications are family-specific and much 446

more frequent than duplications of any other SMC5/6 complex members in plant 447

genomes (reviewed in Diaz and Pecinka 2018). Our data from Arabidopsis and 448

published data from humans (Hudson et al., 2011) suggest that at least some of these 449

duplicated copies differ in their expression domains. We found that both NSE4A and 450

NSE4B can interact with the core subunits SMC5 and NSE3, but not with NSE1, with 451

the latter two representing members of the NSE1-NSE3-NSE4 subcomplex (Palecek 452

and Gruber, 2015). However, in spite of extensive optimization, we did not detect 453

interactions of the NSE4 proteins with SMC6B. This interaction is very likely to exist in 454

Arabidopsis, but seems particularly difficult to confirm as indicated by previous studies 455

in S. cerevisiae and S. pombe (Palecek et al., 2006; Duan et al., 2009; J. Palecek, 456

pers.communication). This is possibly caused by a steric hindrance due to the specific 457

conformation of SMC6 and NSE4 proteins or the absence of an activating and/or 458

stabilizing component. 459

A strong nse4a mutation was homozygous lethal and self-pollinated heterozygotes 460

showed 28.8% seed abortion. This resembles the phenotypes of smc5, nse1, nse3, and 461

asap1 mutants and the sm6a smc6b double mutant, which show embryonic or cotyledon 462

stage seedling death in Arabidopsis (Watanabe et al., 2009; Xu et al., 2013; Yan et al., 463

2013; Li et al., 2017). However, we also found a hypomorphic nse4a-2 allele, which 464

likely produces a protein with a modified C-terminus. This allele alleviates the problem 465

of homozygous lethality encountered in the loss-of-function allele nse4a-1, thereby 466

enabling the analysis of NSE4A functions during plant development and genotoxic 467

21

stress. Its phenotypes partially resemble those of HPY2 and SNI1 mutants, which 468

survive but are strongly affected in development and fertility (Li et al., 1999; Huang et 469

al., 2009; Ishida et al., 2009). 470

471

NSE4A is involved in sporogenesis, gametogenesis, and seed development 472

We observed prominent and dynamic expression of NSE4A during Arabidopsis 473

reproductive development. In the male gametophyte, NSE4A was expressed in the 474

generative cell lineage but absent in the vegetative cell. This is consistent with the 475

observation that the sperm nucleus is rich in the components of active chromatin 476

control, while the vegetative nucleus has lost multiple repressive chromatin 477

modifications and will no longer divide (Schoft et al., 2009; Abdelsamad and Pecinka, 478

2014; Slotkin et al., 2009). However, the function of NSE4A in pollen development 479

remains unknown. Possibly, NSE4A secures a faster or more accurate response, which 480

is not detected under laboratory conditions, upon environmental challenges affecting 481

genome integrity in the germline. 482

NSE4A is also broadly expressed in ovule primordia, with a notable accumulation 483

in the female meiocyte. Thus, besides its role in male meiosis (Liu et al., 2014), the 484

SMC5/6 complex may play a role during female meiosis, possibly in the process of 485

DNA replication, meiotic recombination, or DNA damage repair. During embryo sac 486

development and early seed development, NSE4A was expressed in synergids and the 487

central cell, and later in the embryo and the syncytial and chalazal endosperm. NSE4A 488

expression at these stages may be interpreted as a functional requirement for genome 489

integrity safeguarding processes, which involve DNA repair as a consequence of the 490

challenges posed by rapid DNA replication and chromatin dynamics in these tissues 491

(Baroux et al., 2007; Baroux and Autran, 2015). Genome integrity is necessary to 492

ensure the proper differentiation and functioning of the progeny and to avoid the 493

propagation of genetic mutations. In addition, but not exclusively, the high levels of 494

NSE4A in the syncytial endosperm may play a role in the detoxification of 495

endogenously occurring replication-derived toxic DNA structures. DNA replication 496

produces a high frequency of inter-twining between nascent chromatids, DNA 497

supercoils, and X-shaped toxic DNA replication intermediates, which all require (to 498

different extents) SMC5/6 functions for resolution (Jeppsson, Carlborg, et al. 2014; 499

Menolfi et al. 2015; reviewed in Diaz and Pecinka 2018). 500

22

While SMC5/6 null mutations lead to early seed abortion, the hypomorphic 501

nse4a-2 mutant produced large glossy seeds with liquid endosperm, which turned 502

brown at later stages and aborted. Seed phenotypes similar to nse4a-1 or nse4a-2 were 503

reported for nse1, nse3, and mms21/hpy2 mutants (Liu et al., 2014; Li et al., 2017). 504

Studies in S. cerevisiae revealed that the SMC5/6 complex is loaded by the SCC1 505

subunit of the cohesin complex to specific sites during DNA replication (Jeppsson et al., 506

2014a). This could explain the similarity of SMC5/6 and cohesin mutant seed 507

phenotypes, and indicates that both complexes cooperate during seed development. This 508

may be supported by the identification of cohesin, and also condensin, mutants in a 509

screen focusing on aberrant seed development (Liu et al., 2002; Tzafrir et al., 2002) and 510

underlines the importance of maintaining genome stability during seed development 511

(reviewed in Diaz and Pecinka, 2017). 512

513

NSE4A but not NSE4B is required for resistance to genotoxic stress 514

The functions of the SMC5/6 complex are widely associated with the maintenance of 515

genome stability (Kegel and Sjögren, 2010; Wu and Yu, 2012; Jeppsson et al., 2014b); 516

however, it was not clear which of the Arabidopsis NSE4 paralogs confers this function. 517

We observed activation of NSE4A, but not NSE4B, in response to genotoxic treatments 518

with drugs inducing various types of DNA damage. In addition, the viable and 519

phenotypically almost wild-type nse4a-2 plants were hypersensitive to the cytidine 520

analog zebularine and the alkylating agent MMS, but not to other treatments. Lack of 521

sensitivity to bleocin, MMC, and HU could be caused by the fact that the mutation we 522

analyzed is not a complete loss-of-function allele and/or that such damages can be 523

processed by SMC5/6-independent pathways. We have previously shown that smc6b 524

mutants are hypersensitive to zebularine-induced damage (Liu et al., 2015). This 525

suggests that the SMC5/6 complex is essential for detoxification from complex toxic 526

structures, such as zebularine-induced DNA damage. DNA repair in response to 527

zebularine treatment is mediated both by ATM and ATR kinases (Liu et al., 2015), 528

which are known to phosphorylate proteins at SQ/TQ motifs (Awasthi et al., 2015). 529

NSE4A contains two adjacent TQ motifs at amino acids 361 to 365 (TQDTQ), which 530

makes it a good candidate for a direct target of phosphorylation by ATM and/or ATR. 531

Recent studies from non-plant models suggest that the SMC5/6 complex acts as 532

an ATP-dependent inter-molecular linker, which helps resolving toxic DNA structures 533

at late-replicating sites, and also prevents recombination between non-homologous 534

23

sequences (Chiolo et al., 2011; Kanno et al., 2015; Menolfi et al., 2015). In Arabidopsis, 535

the SMC5/6 complex promotes the association of sister chromatids and is required for 536

normal levels of homologous recombination (Mengiste et al., 1999; Hanin et al., 2000; 537

Watanabe et al., 2009; Yuan et al., 2014). In addition to its role in somatic DNA 538

damage repair, there is emerging evidence that the SMC5/6 complex also plays a role in 539

immune responses (Yan et al., 2013) and meiosis (Yuan et al., 2014). Our data 540

indirectly support a meiotic role of NSE4A as it strongly accumulates in female 541

meiocytes. However, the exact molecular mechanism of genome maintenance by the 542

SMC5/6 complex remains unknown. 543

544

NSE4B and NSE4A have primarily diversified transcriptionally and NSE4B is not 545

responsive to DNA damage 546

In Arabidopsis, the functions of NSE4B are less clear than those of NSE4A. NSE4B 547

single mutants are morphologically indistinguishable from the wild type and do not 548

worsen the phenotype of a weak nse4a mutant. We found that NSE4B is silenced 549

throughout most of development, except for a small domain in the root apical meristem, 550

leaf stipules, and the embryo up to the globular stage. Based on the results of in silico 551

analyses, which revealed an extensive coverage of the NSE4B locus by histone H3 552

lysine 27 tri-methylation, we hypothesize that NSE4B is controlled by the Polycomb 553

Repressive Complex 2 (reviewed in Mozgova and Hennig 2015). To explore NSE4B’s 554

function in the non-silenced state, we swapped its promoter with that of NSE4A and 555

tested whether NSE4B expressed in the pattern of NSE4A can complement the nse4a 556

phenotypes. The seed abortion phenotype was fully complemented, but we found only a 557

partial rescue under DNA damaging conditions. This points to the dual function of the 558

SMC5/6 complex described in budding yeast (Menolfi et al., 2015): a DNA damage-559

independent function during DNA replication and a DNA damage-dependent function 560

in DNA repair. Both NSE4A and NSE4B seem capable of performing the first function, 561

while DNA damage repair can be done only by NSE4A in Arabidopsis. 562

563

564

565

24

METHODS 566

Plant Material 567

A. thaliana wild type and mutants were in the Col-0 background: nse4a-1568

(SALK_057130), nse4a-2 (GK-768H08), nse4b-1 (SAIL_296_F02), nse4b-2 (GK-569

175D10), smc6b-1 (SALK_ SALK_101968C), hpy2-2 (SAIL_77_G06), atr-2 570

(SALK_032841C), wee1-1 (GK-270E05) and lig4-2 (SALK_044027C). We also used a 571

cyclin-GUS line containing the ProCYCB1;1:CYCB1;1:GUS construct (Colón-572

Carmona et al., 1999) and N1DC1 no. 11 (B11) line containing intramolecular type of 573

HR substrate (Puchta et al., 1995). For promoter reporter constructs, regions 18943545 574

to 18941640 and 7260588 to 7258919 bp upstream of the NSE4A and NSE4B 575

transcription start sites, respectively, were PCR-amplified, cloned into pDONOR221, 576

and recombined into the binary Gateway vector pGWB553 containing the uidA gene 577

encoding β-glucuronidase. The final plasmids were transformed into Agrobacterium 578

tumefaciens strain GV3101 and then into Arabidopsis Col-0 using the floral dip method 579

(Zhang et al., 2006). T1 generation seeds were screened on ½ Murashige and Skoog 580

(½MS) plates containing hygromycin B 25 µg/L (Duchefa Biochemie) and resistant 581

plants were transferred to soil. T2 populations with approximately 75% resistant 582

seedlings, indicating single locus T-DNA insertions, were considered for further 583

analyses. For promoter swap experiments, the NSE4A promoter and genomic region of 584

NSE4B were PCR-amplified and cloned into the pGWB550 vector by MultiSite Cloning 585

Gateway (Thermo Fisher Scientific). Plant transformation and selection was carried out 586

as described above, with the exception that the construct was transformed into the 587

nse4a-2 background. To construct the NSE4A-fluorescent protein translational fusion, 588

the NSE4A promoter, coding sequence, terminator, VENUS N-terminal tag, and a 589

BASTA resistance cassette were cloned using Gibson assembly (New England Biolabs) 590

into pGGA000, pGGC000, pGGE000, pGGB000, and pGGF000, respectively, to 591

generate entry clones. The Greengate cloning reaction was performed as described 592

(Lampropoulos et al., 2013), and the multi entry cassette was assembled into the 593

pAGM4723 backbone. nse4a-2 mutant plants were transformed with this construct as 594

described above. For nse4a-2 complementation analysis, the NSE4A promoter and 595

genomic region of NSE4A were PCR-amplified and cloned into the pGWB550 vector by 596

MultiSite Cloning Gateway (Thermo Fisher Scientific). Plant transformation and 597

screening of transformants was carried out exactly as for the promoter swap experiment. 598

25

Plants were emasculated approximately 48 h prior to pollination in crossing 599

experiments. 600

601

Phylogenetic analysis and shadowing 602

NSE4 protein sequences were retrieved from NCBI and Phytozome (Supplemental 603

Table 1). The protein alignment was performed using the MUSCLE algorithm (Edgar, 604

2004), and the resulting alignment was submitted to Gblocks (Castresana, 2000). 605

Curation and selection of aligned blocks was performed in Gblocks using less stringent 606

parameters. Bootstrap probabilities for each node were calculated with 100 replicates. 607

Original sequences, alignments, and blocks are provided as Supplemental Data Sets 1, 608

2, and 3, respectively. 609

Promoter sequences from all analyzed species were retrieved from Phytozome 610

(Supplemental Table 2). Promoter regions of NSE4A and NSE4B were submitted 611

individually to mVISTA (Frazer et al., 2004) and sequence conservation was calculated 612

using LAGAN program (Brudno et al., 2003). A. thaliana sequences were used as 613

references for pairwise comparisons (Supplemental Figure 2). 614

615

Plant growth conditions and drug treatments 616

For genotyping, crossing and seed production plants were grown in 7 x 7 cm pots filled 617

with peat bog in a climatic chamber under controlled long day conditions (16 h with ca. 618

200 μmol m-2 s-1 light intensity and 21°C during day; 8 h at 19°C during night) with 619

standard 70% humidity. 620

For in vitro experiments, sterilized seeds were evenly spread on sterile ½ MS 621

medium with or without zebularine (Sigma-Aldrich), MMC (Duchefa Biochemie), 622

bleocin (Calbiochem) and HU (Sigma-Aldrich) in concentrations specified in the text 623

and grown at 16 h 150 μmol m-2 s-1 light : 8 h dark at 21°C. Seven-day-old plants were 624

used for root length measurements. For MMS experiment, sterilized seeds were grown 625

in ½ MS medium for 5 days and then transferred to liquid ½ MS medium with and 626

without 100 ppm MMS, and grown for 26 days. Roots from 20 to 25 seedlings per 627

genotype were straightened and in total three replicates were performed. For RNA-628

sequencing, seeds were germinated on drug-free ½ MS solid medium and 9-day-old 629

plants were carefully transferred to liquid ½ MS medium with or without 20 μM 630

zebularine. After 24 h, plants were washed with drug-free liquid ½ MS medium, their 631

26

leaves, hypocotyl and roots were removed and shoot apices were flash-frozen in liquid 632

nitrogen for later use. 633

634

Nucleic acid isolation, cDNA synthesis, and PCR 635

For DNA isolation, leaf material of plants at the rosette stage was harvested and DNA 636

was isolated using the DNeasy Plant Mini Kit (QIAGEN), following the manufacturer’s 637

instructions. For RNA isolation, floral buds were collected, shock frozen in liquid 638

nitrogen, and kept at -80°C until use. Total RNA isolation was performed with QIAzol 639

(QIAGEN) and the RNA integrity was assessed by formaldehyde agarose gel 640

electrophoresis. cDNA synthesis was performed from 1 µg of total RNA as starting 641

material, using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher 642

Scientific) with oligo dT primers according to manufacturer’s instructions. Primers used 643

in this study are provided in Supplemental Tables 7 and 8. For 3´RACE PCR, we 644

performed in total four nested PCR reactions using the primer combinations listed in 645

Supplemental Table 8. The first PCR was performed using a 1/100 dilution of cDNA 646

synthesized from the nse4a-2 mutant. Afterwards, the PCR product was gel purified and 647

used for the subsequent nested PCR reaction. This step was repeated until the fourth 648

reaction. PCR product obtained from the fourth reaction was cloned into the pJET1.2 649

vector and sequenced. 650

651

RNA-sequencing and microarray analysis 652

RNA for RNA-sequencing was isolated using RNeasy Plant Mini kit (QIAGEN) with 653

additional on column DNAse I digestion according to manufacturer’s instructions. RNA 654

sequencing was performed with two biological replicates per experimental point. The 655

libraries were prepared from 1 μg total RNA with RNA integrity number >7.8 656

(Bioanalyzer; Agilent) using TruSeq type RNA kit (Illumina) at the Cologne Genome 657

Centre and sequenced as 100-bp single-end reads on HiSeq2500 (Illumina) instrument. 658

Reads were trimmed and quality filtered with FAST-X tools 659

(http://hannonlab.cshl.edu/fastx_toolkit/). This yielded an average of 18.5 million high-660

quality reads per library. The reads were mapped to the TAIR10 Arabidopsis reference 661

genome using Tophat2 (Kim et al., 2013) with default settings. The coverage of 662

individual genes was retrieved with the Qualimap from the set of uniquely mapped 663

reads and significance (adjusted P value < 0.05) of mRNA level changes estimated with 664

the DESeq package (Anders and Huber, 2010). Publicly available sni1-1 Affymetrix 665

27

Arabidopsis ATH1 GeneChip array data (NASC experiment ID 389, slides 20561-666

20566; GEO Series: GSE6827; Mosher et al., 2006) were analyzed using rma protocol 667

with Bioconductor in R. Venn diagrams were drawn using BioVenn online tool 668

(http://www.biovenn.nl/). 669

670

GUS histochemical staining 671

The staining protocol was adapted according to different tissues. Vegetative tissues 672

were stained as described (Liu et al., 2015). Inflorescences were dissected under a Leica 673

MZ16FA stereomicroscope, fixed for 30 min in ice-cold 4% formaldehyde in 1× PBS 674

buffer, washed for 3×5 min in 1× PBS, and infiltrated with GUS staining solution 675

(Stangeland and Salehian, 2002) under vacuum. After 10-15 min the vacuum was 676

released and samples were incubated at 37°C for 3 days, followed by overnight clearing 677

in ethanol 70%. Subsequently, inflorescences were rinsed with water and mounted in 678

petri dishes containing agarose and water. For staining of ovules and young seeds, 679

developing siliques were first opened and fixed in 90% cold acetone at -20°C for 45 680

min. Afterwards, they were rinsed 3× with 100 mM phosphate buffer, transferred to 681

GUS staining solution, vacuum infiltrated for 5 min and stained at 37°C for 48 h. After 682

staining, pistils and siliques were quickly rinsed with phosphate buffer and mounted in 683

8:2:1 chloral hydrate solution. In order to avoid loss of signal when we observed weak 684

GUS staining, we performed a less severe clearing. We dissected pistils and 685

immediately transferred them to GUS solution. Staining of ovules was performed as 686

described (Vielle-Calzada et al., 2000). After clearing, mounted ovules where 687

immediately imaged using a Zeiss microscope. For GUS and DAPI co-staining of 688

pollen grains, flowers were opened and fixed in cold 3:1 ethanol to acetic acid for 30 689

min. Afterwards, they were rinsed 3x with phosphate buffer, infiltrated with GUS 690

staining solution for 10-15 min and stained for 48 hours at 37°C in dark. Next, flowers, 691

GUS stained anthers were dissected, rinsed with phosphate buffer, transferred to a 692

microscopic slide, further dissected with a needle in DAPI solution (0.4 ug/ml DAPI, 693

0.1 M sodium phosphate buffer pH 7, 0.1% triton-x100 and 1 mM EDTA), covered 694

with a coverslip and then used for microscopy. 695

696

Hoyer´s clearing 697

Clearing of seeds was performed as described by (Liu and Meinke, 1998). 698

699

28

Cell cycle arrest 700

The double homozygous nse4a-2 ProCYCB1;1:CYCB1;1:GUS and nse4b-2 701

ProCYCB1;1:CYCB1;1:GUS plants were grown for 5 d in liquid ½ MS medium, 702

transferred to liquid ½ MS supplemented with 10 µM zebularine for 0, 1, 3, 6, 12, 24, or 703

48 h, GUS-stained overnight, cleared in 70% ethanol, and imaged using a Leica 704

MZ16FA stereomicroscope. 705

706

Confocal Microscopy 707

For cell death analysis, seeds from transgenic lines were grown on vertically positioned 708

plates with ½ MS medium for 4 days, and then transferred for 1 day to liquid ½ MS 709

medium with 20 µM zebularine. Seedlings were stained with 10 μg mL-1 of PI solution 710

(Sigma) for 3 min, followed by a rinsing step with sterilized water, and were placed on 711

slides in a drop of water and then evaluated using the Zeiss laser scanning confocal 712

microscope LSM700. For subcellular localization of NSE4A-VENUS in roots, 713

transgenic lines expressing ProNSE4A:VENUS:NSE4A:TerNSE4A were grown for 5 714

days in either solid ½ MS or ½ MS supplemented with 10 µM zebularine. Afterwards, 715

seedlings were stained with PI, following the same procedure as above, and imaged 716

with a Leica TCS SP8 confocal microscope (Leica Microsystems). For imaging of 717

ovules, pistils were quickly dissected in a drop of water and ovules from different stages 718

mounted on a slide with a drop of water and placed on ice. After few minutes 719

preparations were observed using a Leica TCS SP8 confocal microscope (Leica 720

Microsystems) 721

722

Yeast-two-hybrid assay and bimolecular fluorescence complementation 723

The full-length coding sequences of Arabidopsis SMC5, SMC6A, SMC6B, NSE1, and 724

NSE3 were PCR-amplified from cDNA. SMC5, SMC6A and SMC6B were cloned via 725

restriction digest (Supplemental Table 7) into the vector pGADT7 (Clontech), while 726

NSE1 and NSE3 were cloned into the gateway compatible vector pGADT-GW (Lu et al., 727

2010) to produce a protein fusion with the GAL4 DNA AD in N-terminal orientation. In 728

order to produce a protein fusion with the GAL4 DNA BD, the SMC5, NSE4A and 729

NSE4B PCR fragments were cloned via restriction (Supplemental Table 7) digest into 730

pGBKT7 and NSE1 and NSE3 where cloned via gateway into pGBKT7-GW (Lu et al., 731

2010). In order to avoid negative results due to interference of BD or AD domain with 732

possible interactors, all genes were cloned into both C-terminal pGBKCg and pGADCg 733

29

Y2H vectors, to produce C-terminally tagged GAL4 AD and BD fusion proteins, 734

respectively, with exception of NSE4B, which was only cloned into the pGADCg 735

vector. The hinge and fragments of coils of SMC5 (corresponding to amino acids 415 – 736

699), SMC6A (amino acids 367 – 670), and SMC6B (amino acids 358 – 691) were 737

cloned into the pGBKCg and pGADCg vectors to test for interaction with the core 738

subunits. The GAL4-based interaction was tested in the yeast strain AH109 (Clontech). 739

Co-transformed yeast strains were selected on synthetic defined (SD)/–Leu/–Trp 740

medium. Protein–protein interactions were tested using stringent (SD/–Leu/–Trp/–His) 741

selection medium supplemented with defined concentrations of 3-AT (Supplemental 742

Table 4). The interaction between pGADT7-T and pBKT7-53 was used as the positive 743

control, and that between pGADT7-T and pBKT7-LamC was used as the negative 744

control. For BiFC we used the same coding sequences as for the Y2H experiments. The 745

SMC5, SMC5 (hinge), and NSE3 sequences were cloned into pBATL-nYFP, and NSE4A, 746

NSE4B, NSE1, and SMC6B hinge sequences were cloned into pBaTL-cYFP. Both 747

plasmids produce C-terminal fusion proteins. N. benthamiana leaves were transformed 748

for transient expression as described (Tian et al., 2011). YFP fluorescence was observed 749

using the Zeiss LSM 700 confocal microscope. 750

751

Co-immunoprecipitation and localization assays 752

Here we used the same entry clones as for Y2H and BiFC assays. Hinge SMC5 was 753

cloned into pGWB560, to produce a C-terminus fusion with tagRFP protein. Hinge 754

SMC6A, hinge SMC6B, NSE1 CDS, NSE4A CDS and NSE4B CDS were cloned into 755

pGWB541 to produce a C-terminus tagged EYFP proteins. NSE3 CDS was cloned into 756

pGWB611 to produce a C-terminus FLAG fusion protein. To test interactions of SMC5 757

with NSE4s, full length SMC5 CDS was cloned into pGWB561 to produce a N-terminus 758

tagRFP fusion, while both NSE4A and NSE4B CDS were cloned into pGWB542 to 759

produce N- and C-terminus tagged EYFP fusion proteins. As a negative control we used 760

pSY1, containing GFP CDS driven by 35S promoter. Afterwards the expression clones 761

were transformed into A. tumefaciens GV3101. 762

Fluorescent or epitope tag-conjugated proteins were transiently expressed in N. 763

benthamiana leaves by Agrobacterium-mediated infiltration. Leaves were harvested at 4 764

or 5 days after inoculation and immunoprecipitation was performed with a µMACS 765

GFP Isolation Kit (Miltenyi Biotec). Approximately 1–2 g of plant material was 766

homogenized in 3-fold volume of µMACS lysis buffer containing protease inhibitor 767

30

cocktail for plant cell and tissue extracts (Sigma-Aldrich) and then the lysate was 768

filtered through two layers of miracloth. Afterwards, the lysate was mixed with anti-769

GFP antibody-conjugated magnetic beads and was incubated at 4°C for 60 min with 770

gentle rotation. The GFP-conjugated proteins were purified using a magnetic column 771

according to the manufacturer’s instructions. The immunoprecipitated proteins were 772

analyzed by western blotting using an anti-GFP antibody at 1:1000 (ab290, Abcam), an 773

anti-tagRFP antibody at 1:500 (R10367, Thermo Fisher Scientific), or an anti-FLAG 774

antibody at 1:5000 (3022-100, BioVision) as primary antibodies and HRP-conjugated 775

anti-mouse IgG antibody at 1:15000 (W402, Promega) or HRP-conjugated anti-rabbit 776

IgG antibody at 1:15000 (MB4458, MBL) as secondary antibodies. The 777

chemiluminescences from target proteins of each antibody were visualized with 778

ImmunoStar LD (Wako) on Fusion Pulse system (Vilber Lourmat). 779

For the localization analysis of GFP, EYFP-NSE4A, and EYFP-NSE4B proteins 780

simultaneously expressed with tagRFP-SMC5. Five days after inoculation leaves were 781

observed under the inverted laser confocal microscope FV1200 equipped with a GaAsP 782

detector (Olympus) with excitation wavelength with 473 nm for GFP/EYFP and 559 nm 783

for tagRFP. 784

785

Statistical analysis 786

The values were examined by one-way analysis of variance (ANOVA) and post hoc 787

comparison by Duncan's multiple range test (P ≤ 0.05). Statistical analyses except for 788

RNA-sequencing and microarray analysis were performed using STATISTICA v 13 789

(Stat Soft Inc., USA) software. Fisher test was used to calculate adjusted P-value (q-790

value) in RNA-sequencing and microarray analysis. Raw data and detailed results of the 791

statistical analyses are provided in Supplemental Data Set 6. 792

793

Accession Numbers 794

The following genes names and symbols are associated with this publication: ASAP1 795

(AT2G28130), ATR (AT5G40820), LIG4 (AT5G57160), HPY2 (AT3G15150), NSE1 796

(AT5G21140), NSE3 (AT1G34770), NSE4A (AT1G51130), NSE4B (AT3G20760), 797

SMC5 (AT5G15920), SMC6A (AT5G07660), SMC6B (AT5G61460), SNI1 798

(AT4G18470), WEE1 (AT1G02970). RNA-sequencing reads are deposited in GEO as 799

the study number GSE113310. 800

801

31

Supplemental Data 802

Supplemental Figure 1. Phylogenetic shadowing of NSE4A and NSE4B promoters. 803

Supplemental Figure 2. Characterization of NSE4A mutation in nse4a-2. 804

Supplemental Figure 3. Characterization of nse4b-1 and nse4b-2 mutations. 805

Supplemental Figure 4. Chromatin environment of the NSE4A genomic region. 806

Supplemental Figure 5. Chromatin environment of the NSE4B genomic region. 807

Supplemental Figure 6. Seed phenotypes of NSE4A mutants. 808

Supplemental Figure 7. Mutant sensitivity to hydroxyurea (HU). 809

Supplemental Figure 8. Y2H assays (autoactivation and negative results). 810

Supplemental Figure 9. Whole blots from Co-Immunoprecipitation (Co-IP) assays. 811

Supplemental Table 1. Protein sequences used in the phylogenetic analysis. 812

Supplemental Table 2. Promoter sequences used for the phylogenetic shadowing. 813

Supplemental Table 3. Gene ontology (GO) term analysis of 82 genes up-regulated in 814

nse4a-2 and sni1-1. 815

Supplemental Table 4. 3-AT concentrations used in yeast-two-hybrid experiments. 816

Supplemental Table 5. Punnett square indicating frequencies of genotypes in F2 817

generation of self-pollinated F1 hybrid nse4a-1/nse4a-2 T/0. 818

Supplemental Table 6. Promoter swap rescues nse4a-1 lethality. 819

Supplemental Table 7. Primers used in this study. 820

Supplemental Table 8. Primer combinations used in 3´RACE PCR of NSE4A in nse4a-821

2.822

Supplemental Data Set 1. Protein sequences used to build NSE4 phylogenetic tree 823

(FASTA file). 824

Supplemental Data Set 2. Alignment from the full length NSE4 protein sequences 825

submitted to Gblocks server (FASTA file). 826

Supplemental Data Set 3. Gblocks output. Conserved blocks from NSE4 protein 827

alignment (FASTA file). 828

Supplemental Data Set 4. Transcriptomic study of nse4a-2 under control and 829

zebularine stress conditions. 830

Supplemental Data Set 5. Comparison of nse4a-2 and sni1-1 induced transcriptional 831

changes. 832

Supplemental Data Set 6. Background data for and the results of the statistical 833

analyses. 834

835

32

836

ACKNOWLEDGEMENTS 837

We thank Andreas Finke, Fen Yang and Jan Palecek for helpful discussions, Peter 838

Doerner for the cyclin reporter construct, Barbara Eilts, Regina Gentges and Barbara 839

Piwowarczyk for assistance. This work was supported by funding from the Max Planck 840

Society to A.P., P.P. and P.Y.G., DAAD scholarships to M.D. (A/12/77772) and A.N. 841

(ST21 2015/16), and funds from the University of Zurich to U.G.. H.J. and A.P. were 842

supported by the project CZ.02.1.01/0.0/0.0/16_019/0000827 from the MEYS, Czech 843

Republic. A.P. was further supported by the Purkyně Fellowship from the Czech 844

Academy of Sciences, GAČR grant 19-13848S and INTER-COST grant LTC18026. 845

The work of Matsunaga lab was supported by the MXT/JSPS KAKENHI (15H05955 846

and 15H05962) grants to S.M. 847

848

849

AUTHOR CONTRIBUTIONS 850

A.P. and M.D. designed the research with help from C.B., T.S., U.G. and S.M. on851

specific aspects. M.D., P.P., A.N., T.S., H.J., C.B. and P.Y.G. performed the 852

experiments and analyzed the data. A.P. and M.D. wrote the manuscript with 853

contributions from all authors. All authors approved the final version of this article. 854

855

856

FIGURE LEGENDS 857

858

Figure 1. Basic characterization of NSE4 paralogs. 859

(A) Gene structure of A. thaliana NSE4A and NSE4B with indicated positions of the860

mutations used in this study. Scale bars: 100 bp. 861

(B) Alignment of Arabidopsis NSE4A and NSE4B proteins.862

(C) Phylogenetic tree of NSE4 homologs in plants based on the Maximum Likelihood863

algorithm (See Materials and Methods). Fission yeast NSE4/RAD62 and human NSE4 864

paralogs were used as outgroups. Brassicaceae and Poaceae NSE4 duplications are 865

indicated by the colored squares. Identifiers of the protein sequences used to build the 866

tree are provided as Supplemental Data Set 1. 867

(D-F) Phenotypes of homozygous wild-type (WT), nse4a-2 (4a-2), nse4a-2 868

complemented with ProNSE4A:GenomicNSE4A (4a-2 com4A), nse4b-2 (4b-2), and 869

33

nse4a-2 nse4b-2 (4a-2 4b-2) plants. (D) One-week-old in vitro grown seedlings. Bar = 870

10 mm. (E) Three-week-old plants in soil. Bar = 25 mm. (F) Six-week-old mature 871

plants. Bar = 35 mm. 872

(G-I) Analysis of NSE4A and NSE4B promoter activity using the GUS reporter system. 873

(G) One-week-old plants grown as described in (D). Red arrowheads indicate 874

ProNSE4B:GUS signals in the root meristematic zone and leaf stipules (upper inset). 875

(H) 14-day-old plants grown in in vitro culture. (I) Flowers at developmental stages 10–876

14 (Stg 10–Stg14) (Bowman, 1994). Bars = 500 μm. 877

878

Figure 2. NSE4 expression analysis during pollen, ovule, and seed development. 879

(A) Test for functionality of NSE4-VENUS translational fusion line. Wild-type (WT), 880

nse4a-2 (4a-2) and nse4a-2 complemented with ProNSE4A:NSE4A:VENUS (4A-881

VENUS) were germinated and grown on the control and zebularine-containing media 882

for seven days. Restoration of root growth in 4a-2 NSE4A-VENUS indicates full 883

functionality of the translational fusion protein. 884

(B) The first two columns show DAPI- and GUS-stained pollen of ProNSE4A:GUS 885

(Pro4A:GUS) reporter line. Stage (Stg) 10 corresponds to the microspore, stage 11 to 886

bicellular pollen, stage 12 to tricellular pollen, and stage 14 to mature pollen from open 887

anthers. The last column shows pollen from the 4A-VENUS reporter line. Bar = 5 µm. 888

(C) The ProNSE4B:GUS (Pro4B:GUS) reporter line presented in the same way as in 889

(A). Bar = 5 µm. 890

(D) GUS activity of Pro4A:GUS (left) and Pro4B:GUS (right) from ovule primordia to 891

early post-fertilization. Stages 10, 11 and 12 to 14 show ovule primordia, the nucellus, 892

and developing the embryo sac, respectively. Bars = 50 µm. 893

(E) 4A-VENUS signals at the same stages as described in (C). In the ovule primordia of 894

stage 11, the megaspore mother cell (MMC) is almost free of 4A-VENUS signal 895

(arrowheads). However, its expression is greatly increased in the female meiocyte of 896

stage 11 (arrowhead). Bar = 10 µm. 897

(F) GUS activity driven by the NSE4A and NSE4B promoters at the indicated hours 898

after pollination (HAP). Reporter lines were pollinated with their own pollen 48 h after 899

emasculation. e = embryo, ce = chalazal endosperm. Bars = 50 µm. 900

(G) Accumulation of 4A-VENUS in nuclei of globular, heart, torpedo, and bent 901

cotyledon stage embryos and syncytial endosperm 72 HAP. Left images represent 902

34

differential interference contrast (DIC) and the right ones show the VENUS signal. Bars 903

= 50 µm. 904

905

Figure 3. NSE4A is necessary for seed development. 906

(A) Seed phenotypes in wild-type (WT), heterozygous self-pollinated NSE4A/nse4a-1907

(4a-1), homozygous nse4a-2 (4a-2), homozygous nse4a-2 complemented with genomic 908

NSE4A locus (4a-2 com4A), nse4b-1 (4b-1), nse4b-2 (4b-2), and homozygous 4a-2 4b-2 909

double mutant plants. Abnormally developing seeds are indicated by white arrowheads. 910

Non-developing ovules are indicated by white asterisks. 911

(B) Quantification of aborted seeds in the genotypes listed in (A). Error bars indicate912

standard deviation between means of three biological replicates. Each replicate was 913

represented by one plant from which 140 to 300 seeds were analyzed. All plants were 914

grown at the same time. Values marked with the same letter do not differ according to 915

Duncan’s multiple range test (P ≤ 0.05). 916

(C) Equally old cleared WT, pale self-pollinated NSE4A/nse4a-1 (4a-1), and large917

nse4a-2 (4a-2) seeds. Additional nse4-2 seeds are shown in Supplemental Figure 6A. 918

Embryos were outlined by black dashed lines for easier visibility. Bars = 50 µm. 919

920

Figure 4. NSE4A is induced upon DNA damage stimulus. 921

(A) Transcriptional response of the ProNSE4A and ProNSE4B promoters after seven922

days of treatment with 10 µM zebularine (Zeb) in the root apical meristem (RAM) and 923

differentiated root (DR) section with emerging lateral roots (LR). Error bars = 50 µM. 924

(B) 4A-VENUS (nse4a-2 ProNSE4A:NSE4A:VENUS) accumulation in the RAM under925

control conditions and with 10 µM zebularine. Error bars = 50 µM. 926

(C) Transcriptional response of the ProNSE4A and ProNSE4B promoters to 25 nm927

bleocin treatment. Each composite image shows (from top to down and from left to 928

right): cotyledons and the first pair of true leaves, main root apical meristem, detail of 929

the first pair of true leaves, differentiated root zone. 930

931

Figure 5. NSE4A is involved in somatic DNA damage repair. 932

(A) Sensitivity to genotoxic stress. WT, nse4a-2 (4a-2), nse4b-1 (4b-1), nse4b-2 (4b-2),933

nse4a-2 nse4b-2 (4a-2 4b-2), nse4a-2 complemented with genomic NSE4A locus (4a-2 934

com4A), smc6b-1, atr-2, lig4-2 and wee1-1 plants were germinated and maintained for 935

35

one week on 10 µM zebularine, 50 nM bleocin, 10 µM mitomycin C (MMC) or 1 mM 936

hydroxyurea (HU). Bar = 10 mm. 937

(B) Quantitative data for (A) calculated as the relative root length under drug versus938

control conditions. Error bars represent standard deviation between means of three 939

biological replicates. The replicates were grown on separate screening plates and each 940

contained at least 25 plants. Values marked with the same letter do not differ according 941

to Duncan’s multiple range test (P ≤ 0.05). 942

(C) Sensitivity to methyl methanesulfonate (MMS). Representative phenotypes of WT,943

4a-2, 4b-2, 4a-2 4b-2 double mutant, and hpy2-2 plants grown for one week in control 944

liquid medium and then for three weeks in control and 100 ppm MMS containing 945

media. Bar = 10 mm. 946

(D) Analysis of DNA damage repair by homologous recombination using B11 reporter947

line in WT, 4a-2 and 4b-2 backgrounds. Identically colored columns represent 948

individual lines obtained from segregating hybrid populations. Error bars represent 949

mean of three biological replicates, each with at least 30 plants. Values marked with the 950

same letter do not differ according to Duncan’s multiple range test (P ≤ 0.05). 951

(E) Cell death assay. Propidium iodide-stained roots from living Arabidopsis seedlings952

treated without (Mock) and with 20 µM zebularine for 24 h. 953

(F) Meristem size estimation. Plants from (E) were used to estimate the number of cells954

within the root apical meristem (indicated by white arrowheads). Error bars in graph 955

indicate standard deviation among primary roots from five to twelve analyzed plants per 956

each genotype. All plants were grown at the same time. Values marked with the same 957

letter do not differ according to Duncan’s multiple range test (P ≤ 0.05). 958

(G) G2-M cell cycle progression in nse4a-2 and nse4b-2 analyzed by CycB1;1-GUS959

(ProCycB1;1:CycB1;1:GUS) after exposure to 10 µM zebularine for the indicated 960

number of hours (h). 961

962

Figure 6. Transcriptome analysis of nse4a-2 plants. 963

(A) Venn diagrams of genes significantly (DESeq, adjusted P < 0.05) up- and down-964

regulated in dissected shoot apices of 20 µM zebularine-treated wild type (WT 965

zebularine/WT mock), mock-treated nse4a-2 (nse4a-2 mock/WT mock) and 20 µM 966

zebularine-treated nse4a-2 (nse4a-2 zebularine/nse4a-2 mock). The data are based on 967

two RNA-sequencing replicates. 968

36

(B) mRNA abundance of DNA damage repair marker genes expressed as fragments per 969

kilobase per million of reads (FPKM) based on data shown in (A). Asterisks and dashes 970

indicate statistically significant and non-significant, respectively, differences between 971

groups indicated by horizontal bar in DESeq (adjusted P value < 0.05). 972

(C) Venn diagrams of significantly up- and down-regulated genes in nse4a-2 (see A)973

and sni1-1 (sni1-1 mock/WT mock; ATH1 expression microarrays, adjusted P < 0.05) 974

plants. 975

(D) Gene ontology (GO) term analysis of 82 genes significantly up-regulated in both976

nse4a-2 and sni1-1 (see C) using agriGO v2.0. Top ten GO term categories are shown 977

as input relative to Arabidopsis genomic background/reference. The full list of 978

significant GO terms is available in Supplemental Table 3. 979

(E) Examples of significantly (DESeq, adjusted P value < 0.05) up-regulated defense-980

related genes in dissected shoot apices of mock-treated nse4a-2 plants. 981

982

Figure 7. Analysis of protein-protein interactions. 983

(A) Yeast-two-hybrid (Y2H) assays. T+53 = positive control and T + lamC = negative984

control; BD = binding domain and AD = activation domain. Domain position 985

before/after the gene name indicates N- or C-terminal fusions, respectively. 986

Autoactivation controls, negatively tested combinations, and used 3-AT concentrations 987

are provided in Supplemental Figure 8 and Supplemental Table 4. 988

(B) Bimolecular fluorescence complementation (BiFC) validation of interactions989

indicated by Y2H. Insets show nuclei with positive signals. Scale bars = 50 µm. 990

(C) Co-Immunoprecipitation (Co-IP) and co-localization assays. Right panel displays991

Co-IP analysis. IP = input, i.e. total protein extract; Elu = elution, i.e. proteins collected 992

by GFP trapping. Whole blots are shown in Supplemental Figure 9. Right panel shows 993

changes in EYFP-NSE4A and EYFP-NSE4B localization after addition of SMC5-994

tagRFP. 995

(D) The model of protein-protein interactions within Arabidopsis SMC5/6 complex996

based on Y2H and BiFC (red lines), pull-down (Yan et al., 2013) and Co-IP (green 997

lines) experiments. Negatively tested combinations in Y2H are indicated by gray lines. 998

Interaction between HPY2 and SMC5 was published previously (Xu et al., 2013). 999

1000

Figure 8. Analysis of NSE4B functions. 1001

37

(A) Zebularine hypersensitivity assay. WT, nse4a-2 (4a-2), and nse4a-2 complemented 1002

with ProNSE4A:GenNSE4B:TerNSE4B (4a-2 swap) line 13 were germinated and kept 1003

on control and 10 µM zebularine (Zeb) containing media for one week. Bar = 10 mm. 1004

(B) Quantitative data for root length of zebularine-treated versus control plants as1005

described in (A). Lines 11, 12, 13, and 15 represent independent promoter swap 1006

transgenic lines. Error bars indicate standard deviation between the means from two 1007

biological replicates. Each replicate consisted from at least 20 plants per line grown on 1008

separate screening plates at different times. Values marked with the same letter do not 1009

differ according to Duncan’s multiple range test (P ≤ 0.05). 1010

(C) Analysis of seed development phenotypes in WT, heterozygous NSE4A/nse4a-11011

(4a-1), and 4a-2. The two bottom pictures show homozygous nse4a-1 and nse4a-2 1012

containing homozygous promoter swap line 13 (4a-1 swap and 4a-2 swap). White 1013

arrowheads indicate aberrantly developing seeds and asterisks aborted ovules. 1014

38

(D) Quantification of abortion rates in the genotypes described in (C). Error bars 1015

indicate standard deviation between means of three biological replicates (plants), each 1016

with at least 300 scored seeds. Values marked with the same letter do not differ 1017

according to Duncan’s multiple range test (P ≤ 0.05). 1018

Parsed CitationsAbdelsamad, A. and Pecinka, A. (2014). Pollen-specific activation of Arabidopsis retrogenes is associated with global transcriptionalreprogramming. Plant Cell 26: 3299–3313.

Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Alabert, C. and Groth, A. (2012). Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol 13: 153–167.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Anders, S. and Huber, W. (2010). Differential expression analysis for sequence count data. Genome Biol 11: R106.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Awasthi, P., Foiani, M., and Kumar, A. (2015). ATM and ATR signaling at a glance. J. Cell Sci.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Baroux, C. and Autran, D. (2015). Chromatin dynamics during cellular differentiation in the female reproductive lineage of floweringplants. Plant J 83: 160–176.


Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., and Grossniklaus, U. (2007). The triploid endosperm genome of Arabidopsis adopts apeculiar, parental-dosage-dependent chromatin organization. Plant Cell 19: 1782–1794.


Bowman, J.L. (1994). Ovules. In Arabidopsis, pp. 297–331.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Brudno, M., Do, C.B., Cooper, G.M., Kim, M.F., Davydov, E., Program, N.C.S., Green, E.D., Sidow, A., and Batzoglou, S. (2003). LAGANand Multi-LAGAN: Efficient Tools for Large-Scale Multiple Alignment of Genomic DNA. Genome Res. 13: 721–731.


Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their Use in phylogenetic analysis. Mol. Biol. Evol.17: 540–552.


Chiolo, I., Minoda, A., Colmenares, S.U., Polyzos, A., Costes, S. V, and Karpen, G.H. (2011). Double-strand breaks in heterochromatinmove outside of a dynamic HP1a domain to complete recombinational repair. Cell 144: 732–744.


Colón-Carmona, A., You, R., Haimovitch-Gal, T., and Doerner, P. (1999). Spatio-temporal analysis of mitotic activity with a labile cyclin–GUS fusion protein. Plant J 20: 503–508.


Diaz, M. and Pecinka, A. (2018). Scaffolding for repair: Understanding molecular functions of the SMC5/6 complex. Genes (Basel). 9: 36.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Diaz, M. and Pecinka, A. (2017). Seeds as emerging hotspot for maintenance of genome stability. Cytologia (Tokyo). 82: 467–480.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Duan, X., Yang, Y., Chen, Y.-H., Arenz, J., Rangi, G.K., Zhao, X., and Ye, H. (2009). Architecture of the Smc5/6 complex ofSaccharomyces cerevisiae reveals a unique interaction between the Nse5-6 subcomplex and the hinge regions of Smc5 and Smc6. JBiol Chem 284: 8507–8515.


Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Frazer, K.A., Pachter, L., Poliakov, A., Rubin, E.M., and Dubchak, I. (2004). VISTA: computational tools for comparative genomics.Nucleic Acids Res. 32: W273–W279.

Pubmed: Author and Title

https://www.ncbi.nlm.nih.gov/pubmed?term=Abdelsamad%2C A%2E and Pecinka%2C A%2E %282014%29%2E Pollen%2Dspecific activation of Arabidopsis retrogenes is associated with global transcriptional reprogramming%2E Plant Cell 26%3A 3299%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Abdelsamad,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Pollen-specific activation of Arabidopsis retrogenes is associated with global transcriptional reprogramming&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Pollen-specific activation of Arabidopsis retrogenes is associated with global transcriptional reprogramming&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Abdelsamad,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Alabert%2C C%2E and Groth%2C A%2E %282012%29%2E Chromatin replication and epigenome maintenance%2E Nat Rev Mol Cell Biol 13%3A 153%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Alabert,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin replication and epigenome maintenance&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin replication and epigenome maintenance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Alabert,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Anders%2C S%2E and Huber%2C W%2E %282010%29%2E Differential expression analysis for sequence count data%2E Genome Biol 11%3A R&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Anders,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Differential expression analysis for sequence count data&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Differential expression analysis for sequence count data&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Anders,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Awasthi%2C P%2E%2C Foiani%2C M%2E%2C and Kumar%2C A%2E %282015%29%2E ATM and ATR signaling at a glance%2E J%2E Cell Sci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Awasthi,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=ATM and ATR signaling at a glance.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=ATM and ATR signaling at a glance.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Awasthi,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baroux%2C C%2E and Autran%2C D%2E %282015%29%2E Chromatin dynamics during cellular differentiation in the female reproductive lineage of flowering plants%2E Plant J 83%3A 160%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baroux,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin dynamics during cellular differentiation in the female reproductive lineage of flowering plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin dynamics during cellular differentiation in the female reproductive lineage of flowering plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baroux,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Baroux%2C C%2E%2C Pecinka%2C A%2E%2C Fuchs%2C J%2E%2C Schubert%2C I%2E%2C and Grossniklaus%2C U%2E %282007%29%2E The triploid endosperm genome of Arabidopsis adopts a peculiar%2C parental%2Ddosage%2Ddependent chromatin organization%2E Plant Cell 19%3A 1782%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Baroux,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Baroux,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Bowman%2C J%2EL%2E %281994%29%2E Ovules%2E In Arabidopsis%2C pp%2E 297%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bowman,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Ovules.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Ovules.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Bowman,&as_ylo=1994&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Brudno%2C M%2E%2C Do%2C C%2EB%2E%2C Cooper%2C G%2EM%2E%2C Kim%2C M%2EF%2E%2C Davydov%2C E%2E%2C Program%2C N%2EC%2ES%2E%2C Green%2C E%2ED%2E%2C Sidow%2C A%2E%2C and Batzoglou%2C S%2E %282003%29%2E LAGAN and Multi%2DLAGAN%3A Efficient Tools for Large%2DScale Multiple Alignment of Genomic DNA%2E Genome Res%2E 13%3A 721%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Brudno,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=LAGAN and Multi-LAGAN: Efficient Tools for Large-Scale Multiple Alignment of Genomic DNA&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=LAGAN and Multi-LAGAN: Efficient Tools for Large-Scale Multiple Alignment of Genomic DNA&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Brudno,&as_ylo=2003&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Castresana%2C J%2E %282000%29%2E Selection of conserved blocks from multiple alignments for their Use in phylogenetic analysis%2E Mol%2E Biol%2E Evol%2E 17%3A 540%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Castresana,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Selection of conserved blocks from multiple alignments for their Use in phylogenetic analysis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Selection of conserved blocks from multiple alignments for their Use in phylogenetic analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Castresana,&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Chiolo%2C I%2E%2C Minoda%2C A%2E%2C Colmenares%2C S%2EU%2E%2C Polyzos%2C A%2E%2C Costes%2C S%2E V%2C and Karpen%2C G%2EH%2E %282011%29%2E Double%2Dstrand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair%2E Cell 144%3A 732%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Chiolo,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Chiolo,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Col�n%2DCarmona%2C A%2E%2C You%2C R%2E%2C Haimovitch%2DGal%2C T%2E%2C and Doerner%2C P%2E %281999%29%2E Spatio%2Dtemporal analysis of mitotic activity with a labile cyclin%26%23x2013%3BGUS fusion protein%2E Plant J 20%3A 503%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Colón-Carmona,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Spatio-temporal analysis of mitotic activity with a labile cyclin?GUS fusion protein&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Spatio-temporal analysis of mitotic activity with a labile cyclin?GUS fusion protein&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Colón-Carmona,&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Diaz%2C M%2E and Pecinka%2C A%2E %282018%29%2E Scaffolding for repair%3A Understanding molecular functions of the SMC5%2F6 complex%2E Genes %28Basel&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Diaz,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Scaffolding for repair: Understanding molecular functions of the SMC5/6 complex&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Scaffolding for repair: Understanding molecular functions of the SMC5/6 complex&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Diaz,&as_ylo=2018&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Diaz%2C M%2E and Pecinka%2C A%2E %282017%29%2E Seeds as emerging hotspot for maintenance of genome stability%2E Cytologia %28Tokyo%29%2E 82%3A 467%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Diaz,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Seeds as emerging hotspot for maintenance of genome stability&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Seeds as emerging hotspot for maintenance of genome stability&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Diaz,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Duan%2C X%2E%2C Yang%2C Y%2E%2C Chen%2C Y%2E%2DH%2E%2C Arenz%2C J%2E%2C Rangi%2C G%2EK%2E%2C Zhao%2C X%2E%2C and Ye%2C H%2E %282009%29%2E Architecture of the Smc5%2F6 complex of Saccharomyces cerevisiae reveals a unique interaction between the Nse5%2D6 subcomplex and the hinge regions of Smc5 and Smc6%2E J Biol Chem 284%3A 8507%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Duan,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Architecture of the Smc5/6 complex of Saccharomyces cerevisiae reveals a unique interaction between the Nse5-6 subcomplex and the hinge regions of Smc5 and Smc6&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Architecture of the Smc5/6 complex of Saccharomyces cerevisiae reveals a unique interaction between the Nse5-6 subcomplex and the hinge regions of Smc5 and Smc6&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Duan,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Edgar%2C R%2EC%2E %282004%29%2E MUSCLE%3A multiple sequence alignment with high accuracy and high throughput%2E Nucleic Acids Res 32%3A 1792%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Edgar,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=MUSCLE: multiple sequence alignment with high accuracy and high throughput&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=MUSCLE: multiple sequence alignment with high accuracy and high throughput&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Edgar,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Frazer%2C K%2EA%2E%2C Pachter%2C L%2E%2C Poliakov%2C A%2E%2C Rubin%2C E%2EM%2E%2C and Dubchak%2C I%2E %282004%29%2E VISTA%3A computational tools for comparative genomics%2E Nucleic Acids Res%2E 32%3A W273%26%23x2013%3BW&dopt=abstract

Google Scholar: Author Only Title Only Author and Title

Hanin, M., Mengiste, T., Bogucki, A., and Paszkowski, J. (2000). Elevated levels of intrachromosomal homologous recombination inArabidopsis overexpressing the MIM gene. Plant J. 24: 183–189.


Hirano, T. (2006). At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol 7: 311–322.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hirano, T. (2012). Condensins: universal organizers of chromosomes with diverse functions. Genes Dev 26: 1659–1678.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Huang, L., Yang, S., Zhang, S., Liu, M., Lai, J., Qi, Y., Shi, S., Wang, J., Wang, Y., Xie, Q., and Yang, C. (2009). The Arabidopsis SUMO E3ligase AtMMS21, a homologue of NSE2/MMS21, regulates cell proliferation in the root. Plant J 60: 666–678.


Hudson, J.J.R., Bednarova, K., Kozakova, L., Liao, C., Guerineau, M., Colnaghi, R., Vidot, S., Marek, J., Bathula, S.R., Lehmann, A.R.,and Palecek, J. (2011). Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conservedinteractions between MAGE and EID families. PLoS One 6: e17270.


Ishida, T., Fujiwara, S., Miura, K., Stacey, N., Yoshimura, M., Schneider, K., Adachi, S., Minamisawa, K., Umeda, M., and Sugimoto, K.(2009). SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis. Plant Cell 21: 2284–2297.


Jeppsson, K., Carlborg, K.K., Nakato, R., Berta, D.G., Lilienthal, I., Kanno, T., Lindqvist, A., Brink, M.C., Dantuma, N.P., Katou, Y.,Shirahige, K., and Sjögren, C. (2014a). The chromosomal association of the Smc5/6 complex depends on cohesion and predicts thelevel of sister chromatid entanglement. PLoS Genet 10: e1004680.


Jeppsson, K., Kanno, T., Shirahige, K., and Sjogren, C. (2014b). The maintenance of chromosome structure: positioning andfunctioning of SMC complexes. Nat Rev Mol Cell Biol 15: 601–614.


Kagale, S., Robinson, S.J., Nixon, J., Xiao, R., Huebert, T., Condie, J., Kessler, D., Clarke, W.E., Edger, P.P., Links, M.G., Sharpe, A.G.,and Parkin, I.A.P. (2014). Polyploid evolution of the Brassicaceae during the cenozoic era. Plant Cell 26: 2777–2791.


Kanno, T., Berta, D.G., and Sjögren, C. (2015). The Smc5/6 complex is an ATP-dependent intermolecular DNA linker. Cell Rep 12: 1471–1482.


Kegel, A. and Sjögren, C. (2010). The Smc5/6 complex: More than repair? Cold Spring Harb. Symp Quant. Biol 75: 179–187.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., and Salzberg, S. (2013). TopHat2: accurate alignment of transcriptomes in thepresence of insertions, deletions and gene fusions. Genome Biol. 14: R36.


Kozak, J., West, C.E., White, C., da Costa-Nunes, J.A., and Angelis, K.J. (2009). Rapid repair of DNA double strand breaks in Arabidopsisthaliana is dependent on proteins involved in chromosome structure maintenance. DNA Repair (Amst). 8: 413–419.


Kwak, J.S., Son, G.H., Kim, S.-I., Song, J.T., and Seo, H.S. (2016). Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes FloweringLocus C through its E3 ligase activity. Front Plant Sci 7: 530.


Lampropoulos, A., Sutikovic, Z., Wenzl, C., Maegele, I., Lohmann, J.U., and Forner, J. (2013). GreenGate - A novel, versatile, andefficient cloning system for plant transgenesis. PLoS One 8: e83043.


https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Frazer,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=VISTA: computational tools for comparative genomics&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=VISTA: computational tools for comparative genomics&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Frazer,&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hanin%2C M%2E%2C Mengiste%2C T%2E%2C Bogucki%2C A%2E%2C and Paszkowski%2C J%2E %282000%29%2E Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene%2E Plant J%2E 24%3A 183%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hanin,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hanin,&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hirano%2C T%2E %282006%29%2E At the heart of the chromosome%3A SMC proteins in action%2E Nat Rev Mol Cell Biol 7%3A 311%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hirano,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=At the heart of the chromosome: SMC proteins in action&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=At the heart of the chromosome: SMC proteins in action&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hirano,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hirano%2C T%2E %282012%29%2E Condensins%3A universal organizers of chromosomes with diverse functions%2E Genes Dev 26%3A 1659%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hirano,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Condensins: universal organizers of chromosomes with diverse functions&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Condensins: universal organizers of chromosomes with diverse functions&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hirano,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Huang%2C L%2E%2C Yang%2C S%2E%2C Zhang%2C S%2E%2C Liu%2C M%2E%2C Lai%2C J%2E%2C Qi%2C Y%2E%2C Shi%2C S%2E%2C Wang%2C J%2E%2C Wang%2C Y%2E%2C Xie%2C Q%2E%2C and Yang%2C C%2E %282009%29%2E The Arabidopsis SUMO E3 ligase AtMMS21%2C a homologue of NSE2%2FMMS21%2C regulates cell proliferation in the root%2E Plant J 60%3A 666%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Huang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Huang,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Hudson%2C J%2EJ%2ER%2E%2C Bednarova%2C K%2E%2C Kozakova%2C L%2E%2C Liao%2C C%2E%2C Guerineau%2C M%2E%2C Colnaghi%2C R%2E%2C Vidot%2C S%2E%2C Marek%2C J%2E%2C Bathula%2C S%2ER%2E%2C Lehmann%2C A%2ER%2E%2C and Palecek%2C J%2E %282011%29%2E Interactions between the Nse3 and Nse4 components of the SMC5%2D6 complex identify evolutionarily conserved interactions between MAGE and EID families%2E PLoS One 6%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Hudson,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID families&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID families&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Hudson,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Ishida%2C T%2E%2C Fujiwara%2C S%2E%2C Miura%2C K%2E%2C Stacey%2C N%2E%2C Yoshimura%2C M%2E%2C Schneider%2C K%2E%2C Adachi%2C S%2E%2C Minamisawa%2C K%2E%2C Umeda%2C M%2E%2C and Sugimoto%2C K%2E %282009%29%2E SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis%2E Plant Cell 21%3A 2284%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Ishida,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SUMO E3 ligase HIGH PLOIDY2 regulates endocycle onset and meristem maintenance in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Ishida,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Jeppsson%2C K%2E%2C Carlborg%2C K%2EK%2E%2C Nakato%2C R%2E%2C Berta%2C D%2EG%2E%2C Lilienthal%2C I%2E%2C Kanno%2C T%2E%2C Lindqvist%2C A%2E%2C Brink%2C M%2EC%2E%2C Dantuma%2C N%2EP%2E%2C Katou%2C Y%2E%2C Shirahige%2C K%2E%2C and Sj�gren%2C C%2E %282014a%29%2E The chromosomal association of the Smc5%2F6 complex depends on cohesion and predicts the level of sister chromatid entanglement%2E PLoS Genet 10%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jeppsson,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jeppsson,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Jeppsson%2C K%2E%2C Kanno%2C T%2E%2C Shirahige%2C K%2E%2C and Sjogren%2C C%2E %282014b%29%2E The maintenance of chromosome structure%3A positioning and functioning of SMC complexes%2E Nat Rev Mol Cell Biol 15%3A 601%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Jeppsson,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Jeppsson,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kagale%2C S%2E%2C Robinson%2C S%2EJ%2E%2C Nixon%2C J%2E%2C Xiao%2C R%2E%2C Huebert%2C T%2E%2C Condie%2C J%2E%2C Kessler%2C D%2E%2C Clarke%2C W%2EE%2E%2C Edger%2C P%2EP%2E%2C Links%2C M%2EG%2E%2C Sharpe%2C A%2EG%2E%2C and Parkin%2C I%2EA%2EP%2E %282014%29%2E Polyploid evolution of the Brassicaceae during the cenozoic era%2E Plant Cell 26%3A 2777%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kagale,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Polyploid evolution of the Brassicaceae during the cenozoic era&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Polyploid evolution of the Brassicaceae during the cenozoic era&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kagale,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kanno%2C T%2E%2C Berta%2C D%2EG%2E%2C and Sj�gren%2C C%2E %282015%29%2E The Smc5%2F6 complex is an ATP%2Ddependent intermolecular DNA linker%2E Cell Rep 12%3A 1471%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kanno,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5/6 complex is an ATP-dependent intermolecular DNA linker&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5/6 complex is an ATP-dependent intermolecular DNA linker&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kanno,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kegel%2C A%2E and Sj�gren%2C C%2E %282010%29%2E The Smc5%2F6 complex%3A More than repair%3F Cold Spring Harb%2E Symp Quant%2E Biol 75%3A 179%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kegel,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5/6 complex: More than repair&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5/6 complex: More than repair&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kegel,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kim%2C D%2E%2C Pertea%2C G%2E%2C Trapnell%2C C%2E%2C Pimentel%2C H%2E%2C Kelley%2C R%2E%2C and Salzberg%2C S%2E %282013%29%2E TopHat2%3A accurate alignment of transcriptomes in the presence of insertions%2C deletions and gene fusions%2E Genome Biol%2E 14%3A R&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kim,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kim,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kozak%2C J%2E%2C West%2C C%2EE%2E%2C White%2C C%2E%2C da Costa%2DNunes%2C J%2EA%2E%2C and Angelis%2C K%2EJ%2E %282009%29%2E Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance%2E DNA Repair %28Amst%29%2E 8%3A 413%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kozak,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kozak,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Kwak%2C J%2ES%2E%2C Son%2C G%2EH%2E%2C Kim%2C S%2E%2DI%2E%2C Song%2C J%2ET%2E%2C and Seo%2C H%2ES%2E %282016%29%2E Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes Flowering Locus C through its E3 ligase activity%2E Front Plant Sci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Kwak,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes Flowering Locus C through its E3 ligase activity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes Flowering Locus C through its E3 ligase activity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Kwak,&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lampropoulos%2C A%2E%2C Sutikovic%2C Z%2E%2C Wenzl%2C C%2E%2C Maegele%2C I%2E%2C Lohmann%2C J%2EU%2E%2C and Forner%2C J%2E %282013%29%2E GreenGate %2D A novel%2C versatile%2C and efficient cloning system for plant transgenesis%2E PLoS One 8%3A e&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lampropoulos,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=GreenGate - A novel, versatile, and efficient cloning system for plant transgenesis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=GreenGate - A novel, versatile, and efficient cloning system for plant transgenesis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lampropoulos,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

Li, G., Zou, W., Jian, L., Qian, J., Deng, Y., and Zhao, J. (2017). Non-SMC elements 1 and 3 are required for early embryo and seedlingdevelopment in Arabidopsis. J Exp Bot 68: 1039–1054.


Li, X., Zhang, Y., Clarke, J.D., Li, Y., and Dong, X. (1999). Identification and cloning of a negative regulator of systemic acquiredresistance, SNI1, through a screen for suppressors of npr1-1. Cell 98: 329–339.


Liu, C.-H., Finke, A., Diaz, M., Rozhon, W., Poppenberger, B., Baubec, T., and Pecinka, A. (2015). Repair of DNA damage induced by thecytidine analog zebularine requires ATR and ATM in Arabidopsis. Plant Cell 27: 1788–1800.


Liu, C.-M., McElver, J., Tzafrir, I., Joosen, R., Wittich, P., Patton, D., Van Lammeren, A.A.M., and Meinke, D. (2002). Condensin andcohesin knockouts in Arabidopsis exhibit a titan seed phenotype. Plant J 29: 405–415.


Liu, C.-M. and Meinke, D.W. (1998). The titan mutants of Arabidopsisare disrupted in mitosis and cell cycle control during seeddevelopment. Plant J 16: 21–31.


Liu, M., Shi, S., Zhang, S., Xu, P., Lai, J., Liu, Y., Yuan, D., Wang, Y., Du, J., and Yang, C. (2014). SUMO E3 ligase AtMMS21 is required fornormal meiosis and gametophyte development in Arabidopsis. BMC Plant Biol 14: 153.


Lu, Q., Tang, X., Tian, G., Wang, F., Liu, K., Nguyen, V., Kohalmi, S.E., Keller, W.A., Tsang, E.W.T., Harada, J.J., Rothstein, S.J., and Cui,Y. (2010). Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J 61: 259–270.


Mengiste, T., Revenkova, E., Bechtold, N., and Paszkowski, J. (1999). An SMC-like protein is required for efficient homologousrecombination in Arabidopsis. EMBO J 18: 4505–4512.


Menolfi, D., Delamarre, A., Lengronne, A., Pasero, P., and Branzei, D. (2015). Essential roles of the Smc5/6 complex in replicationthrough natural pausing sites and endogenous DNA damage tolerance. Mol Cell 60: 835–846.


Mosher, R.A., Durrant, W.E., Wang, D., Song, J., and Dong, X. (2006). A comprehensive structure–function analysis of Arabidopsis SNI1defines essential regions and transcriptional repressor activity. Plant Cell 18: 1750–1765.


Mozgova, I. and Hennig, L. (2015). The Polycomb group protein regulatory network. Annu Rev Plant Biol 66: 269–296.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast andefficient maturation for cell-biological applications. Nat. Biotechnol. 20: 87.


Palecek, J., Vidot, S., Feng, M., Doherty, A.J., and Lehmann, A.R. (2006). The Smc5-Smc6 DNA Repair Complex: Bridging of the Smc5-Smc6 heads by the kleisin, Nse4, and non-kleisin subunits. J. Biol. Chem. 281: 36952–36959.


Palecek, J.J. and Gruber, S. (2015). Kite proteins: a superfamily of SMC/kleisin partners conserved across bacteria, archaea, andeukaryotes. Structure 23: 2183–2190.


Paterson, A.H. et al. (2009). The Sorghum bicolor genome and the diversification of grasses. Nature 457: 551–556.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C G%2E%2C Zou%2C W%2E%2C Jian%2C L%2E%2C Qian%2C J%2E%2C Deng%2C Y%2E%2C and Zhao%2C J%2E %282017%29%2E Non%2DSMC elements 1 and 3 are required for early embryo and seedling development in Arabidopsis%2E J Exp Bot 68%3A 1039%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Non-SMC elements 1 and 3 are required for early embryo and seedling development in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Non-SMC elements 1 and 3 are required for early embryo and seedling development in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=2017&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Li%2C X%2E%2C Zhang%2C Y%2E%2C Clarke%2C J%2ED%2E%2C Li%2C Y%2E%2C and Dong%2C X%2E %281999%29%2E Identification and cloning of a negative regulator of systemic acquired resistance%2C SNI1%2C through a screen for suppressors of npr1%2D1%2E Cell 98%3A 329%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Li,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Li,&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C C%2E%2DH%2E%2C Finke%2C A%2E%2C Diaz%2C M%2E%2C Rozhon%2C W%2E%2C Poppenberger%2C B%2E%2C Baubec%2C T%2E%2C and Pecinka%2C A%2E %282015%29%2E Repair of DNA damage induced by the cytidine analog zebularine requires ATR and ATM in Arabidopsis%2E Plant Cell 27%3A 1788%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Liu,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Repair of DNA damage induced by the cytidine analog zebularine requires ATR and ATM in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Repair of DNA damage induced by the cytidine analog zebularine requires ATR and ATM in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C C%2E%2DM%2E%2C McElver%2C J%2E%2C Tzafrir%2C I%2E%2C Joosen%2C R%2E%2C Wittich%2C P%2E%2C Patton%2C D%2E%2C Van Lammeren%2C A%2EA%2EM%2E%2C and Meinke%2C D%2E %282002%29%2E Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype%2E Plant J 29%3A 405%26%23x&dopt=abstract


https://scholar.google.com/scholar?as_q=Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C C%2E%2DM%2E and Meinke%2C D%2EW%2E %281998%29%2E The titan mutants of Arabidopsisare disrupted in mitosis and cell cycle control during seed development%2E Plant J 16%3A 21%26%23x&dopt=abstract


https://scholar.google.com/scholar?as_q=The titan mutants of Arabidopsisare disrupted in mitosis and cell cycle control during seed development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The titan mutants of Arabidopsisare disrupted in mitosis and cell cycle control during seed development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=1998&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Liu%2C M%2E%2C Shi%2C S%2E%2C Zhang%2C S%2E%2C Xu%2C P%2E%2C Lai%2C J%2E%2C Liu%2C Y%2E%2C Yuan%2C D%2E%2C Wang%2C Y%2E%2C Du%2C J%2E%2C and Yang%2C C%2E %282014%29%2E SUMO E3 ligase AtMMS21 is required for normal meiosis and gametophyte development in Arabidopsis%2E BMC Plant Biol&dopt=abstract


https://scholar.google.com/scholar?as_q=SUMO E3 ligase AtMMS21 is required for normal meiosis and gametophyte development in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SUMO E3 ligase AtMMS21 is required for normal meiosis and gametophyte development in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Liu,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Lu%2C Q%2E%2C Tang%2C X%2E%2C Tian%2C G%2E%2C Wang%2C F%2E%2C Liu%2C K%2E%2C Nguyen%2C V%2E%2C Kohalmi%2C S%2EE%2E%2C Keller%2C W%2EA%2E%2C Tsang%2C E%2EW%2ET%2E%2C Harada%2C J%2EJ%2E%2C Rothstein%2C S%2EJ%2E%2C and Cui%2C Y%2E %282010%29%2E Arabidopsis homolog of the yeast TREX%2D2 mRNA export complex%3A components and anchoring nucleoporin%2E Plant J 61%3A 259%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Lu,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Lu,&as_ylo=2010&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Mengiste%2C T%2E%2C Revenkova%2C E%2E%2C Bechtold%2C N%2E%2C and Paszkowski%2C J%2E %281999%29%2E An SMC%2Dlike protein is required for efficient homologous recombination in Arabidopsis%2E EMBO J 18%3A 4505%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mengiste,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=An SMC-like protein is required for efficient homologous recombination in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=An SMC-like protein is required for efficient homologous recombination in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mengiste,&as_ylo=1999&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Menolfi%2C D%2E%2C Delamarre%2C A%2E%2C Lengronne%2C A%2E%2C Pasero%2C P%2E%2C and Branzei%2C D%2E %282015%29%2E Essential roles of the Smc5%2F6 complex in replication through natural pausing sites and endogenous DNA damage tolerance%2E Mol Cell 60%3A 835%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Menolfi,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Essential roles of the Smc5/6 complex in replication through natural pausing sites and endogenous DNA damage tolerance&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Essential roles of the Smc5/6 complex in replication through natural pausing sites and endogenous DNA damage tolerance&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Menolfi,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Mosher%2C R%2EA%2E%2C Durrant%2C W%2EE%2E%2C Wang%2C D%2E%2C Song%2C J%2E%2C and Dong%2C X%2E %282006%29%2E A comprehensive structure%26%23x2013%3Bfunction analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity%2E Plant Cell 18%3A 1750%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mosher,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A comprehensive structure?function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A comprehensive structure?function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mosher,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Mozgova%2C I%2E and Hennig%2C L%2E %282015%29%2E The Polycomb group protein regulatory network%2E Annu Rev Plant Biol 66%3A 269%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Mozgova,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Polycomb group protein regulatory network&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Polycomb group protein regulatory network&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Mozgova,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Nagai%2C T%2E%2C Ibata%2C K%2E%2C Park%2C E%2ES%2E%2C Kubota%2C M%2E%2C Mikoshiba%2C K%2E%2C and Miyawaki%2C A%2E %282002%29%2E A variant of yellow fluorescent protein with fast and efficient maturation for cell%2Dbiological applications%2E Nat%2E Biotechnol&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Nagai,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Nagai,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Palecek%2C J%2E%2C Vidot%2C S%2E%2C Feng%2C M%2E%2C Doherty%2C A%2EJ%2E%2C and Lehmann%2C A%2ER%2E %282006%29%2E The Smc5%2DSmc6 DNA Repair Complex%3A Bridging of the Smc5%2DSmc6 heads by the kleisin%2C Nse4%2C and non%2Dkleisin subunits%2E J%2E Biol%2E Chem%2E 281%3A 36952%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Palecek,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Palecek,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Palecek%2C J%2EJ%2E and Gruber%2C S%2E %282015%29%2E Kite proteins%3A a superfamily of SMC%2Fkleisin partners conserved across bacteria%2C archaea%2C and eukaryotes%2E Structure 23%3A 2183%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Palecek,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Kite proteins: a superfamily of SMC/kleisin partners conserved across bacteria, archaea, and eukaryotes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Kite proteins: a superfamily of SMC/kleisin partners conserved across bacteria, archaea, and eukaryotes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Palecek,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Paterson%2C A%2EH%2E et al%2E %282009%29%2E The Sorghum bicolor genome and the diversification of grasses%2E Nature 457%3A 551%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Paterson,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Sorghum bicolor genome and the diversification of grasses&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Sorghum bicolor genome and the diversification of grasses&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Paterson,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

Pebernard, S., Wohlschlegel, J., McDonald, W.H., Yates, J.R., and Boddy, M.N. (2006). The Nse5-Nse6 dimer mediates DNA repair rolesof the Smc5-Smc6 complex. Mol Cell Biol 26: 1617–1630.


de Piccoli, G., Torres-Rosell, J., and Aragón, L. (2009). The unnamed complex: what do we know about Smc5-Smc6? Chromosom. Res17: 251–263.


Potts, P.R. and Yu, H. (2007). The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins. Nat Struct Mol Biol 14: 581–590.


Puchta, H., Swoboda, P., and Hohn, B. (1995). Induction of intrachromosomal homologous recombination in whole plants. Plant J 7: 203–210.


Räschle, M. et al. (2015). Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. Science 348:1253671.


Schoft, V.K., Chumak, N., Mosiolek, M., Slusarz, L., Komnenovic, V., Brownfield, L., Twell, D., Kakutani, T., and Tamaru, H. (2009).Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin. EMBO Rep 10: 1015–1021.


Schubert, V. (2009). SMC proteins and their multiple functions in higher plants. Cytogenet Genome Res 124: 202–214.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

De Schutter, K., Joubès, J., Cools, T., Verkest, A., Corellou, F., Babiychuk, E., Van Der Schueren, E., Beeckman, T., Kushnir, S., Inzé,D., and De Veylder, L. (2007). Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA IntegrityCheckpoint. Plant Cell 19: 211–225.


Slotkin, R.K., Vaughn, M., Borges, F., Tanurdžić, M., Becker, J.D., Feijó, J.A., and Martienssen, R.A. (2009). Epigenetic reprogrammingand small RNA silencing of transposable elements in pollen. Cell 136: 461–472.


Stangeland, B. and Salehian, Z. (2002). An improved clearing method for GUS assay inArabidopsis endosperm and seeds. Plant Mol.Biol. Report. 20: 107–114.


Stingele, J. and Jentsch, S. (2015). DNA-protein crosslink repair. Nat Rev Mol Cell Biol 16: 455–460.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Tian, G., Lu, Q., Zhang, L., Kohalmi, S.E., and Cui, Y. (2011). Detection of protein interactions in plant using a Gateway compatibleBimolecular Fluorescence Complementation (BiFC) system. J Vis. Exp: 3473.


Torres-Rosell, J., Sunjevaric, I., De Piccoli, G., Sacher, M., Eckert-Boulet, N., Reid, R., Jentsch, S., Rothstein, R., Aragon, L., and Lisby,M. (2007). The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. NatCell Biol 9: 923–931.


Tretyakova, N.Y., Groehler, A., and Ji, S. (2015). DNA-protein cross-links: Formation, structural identities, and biological outcomes. AccChem Res 48: 1631–1644.


Tzafrir, I., McElver, J.A., Liu, C., Yang, L.J., Wu, J.Q., Martinez, A., Patton, D.A., and Meinke, D.W. (2002). Diversity of TITAN Functions inArabidopsis Seed Development. Plant Physiol 128: 38–51.


https://www.ncbi.nlm.nih.gov/pubmed?term=Pebernard%2C S%2E%2C Wohlschlegel%2C J%2E%2C McDonald%2C W%2EH%2E%2C Yates%2C J%2ER%2E%2C and Boddy%2C M%2EN%2E %282006%29%2E The Nse5%2DNse6 dimer mediates DNA repair roles of the Smc5%2DSmc6 complex%2E Mol Cell Biol 26%3A 1617%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Pebernard,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Pebernard,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=de Piccoli%2C G%2E%2C Torres%2DRosell%2C J%2E%2C and Arag�n%2C L%2E %282009%29%2E The unnamed complex%3A what do we know about Smc5%2DSmc6%3F Chromosom%2E Res 17%3A 251%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=de&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The unnamed complex: what do we know about Smc5-Smc6&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The unnamed complex: what do we know about Smc5-Smc6&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=de&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Potts%2C P%2ER%2E and Yu%2C H%2E %282007%29%2E The SMC5%2F6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere%2Dbinding proteins%2E Nat Struct Mol Biol 14%3A 581%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Potts,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Potts,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Puchta%2C H%2E%2C Swoboda%2C P%2E%2C and Hohn%2C B%2E %281995%29%2E Induction of intrachromosomal homologous recombination in whole plants%2E Plant J 7%3A 203%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Puchta,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Induction of intrachromosomal homologous recombination in whole plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Induction of intrachromosomal homologous recombination in whole plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Puchta,&as_ylo=1995&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=R�schle%2C M%2E et al%2E %282015%29%2E Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross%2Dlinks%2E Science&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Räschle,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Räschle,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schoft%2C V%2EK%2E%2C Chumak%2C N%2E%2C Mosiolek%2C M%2E%2C Slusarz%2C L%2E%2C Komnenovic%2C V%2E%2C Brownfield%2C L%2E%2C Twell%2C D%2E%2C Kakutani%2C T%2E%2C and Tamaru%2C H%2E %282009%29%2E Induction of RNA%2Ddirected DNA methylation upon decondensation of constitutive heterochromatin%2E EMBO Rep 10%3A 1015%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schoft,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Induction of RNA-directed DNA methylation upon decondensation of constitutive heterochromatin&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schoft,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Schubert%2C V%2E %282009%29%2E SMC proteins and their multiple functions in higher plants%2E Cytogenet Genome Res 124%3A 202%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Schubert,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=SMC proteins and their multiple functions in higher plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SMC proteins and their multiple functions in higher plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schubert,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=De Schutter%2C K%2E%2C Joub�s%2C J%2E%2C Cools%2C T%2E%2C Verkest%2C A%2E%2C Corellou%2C F%2E%2C Babiychuk%2C E%2E%2C Van Der Schueren%2C E%2E%2C Beeckman%2C T%2E%2C Kushnir%2C S%2E%2C Inz�%2C D%2E%2C and De Veylder%2C L%2E %282007%29%2E Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA Integrity Checkpoint%2E Plant Cell 19%3A 211%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=De&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA Integrity Checkpoint&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Arabidopsis WEE1 Kinase Controls Cell Cycle Arrest in Response to Activation of the DNA Integrity Checkpoint&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=De&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Slotkin%2C R%2EK%2E%2C Vaughn%2C M%2E%2C Borges%2C F%2E%2C Tanurdži�?%2C M%2E%2C Becker%2C J%2ED%2E%2C Feijó%2C J%2EA%2E%2C and Martienssen%2C R%2EA%2E %282009%29%2E Epigenetic reprogramming and small RNA silencing of transposable elements in pollen%2E Cell 136%3A 461%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Slotkin,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Epigenetic reprogramming and small RNA silencing of transposable elements in pollen&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Epigenetic reprogramming and small RNA silencing of transposable elements in pollen&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Slotkin,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Stangeland%2C B%2E and Salehian%2C Z%2E %282002%29%2E An improved clearing method for GUS assay inArabidopsis endosperm and seeds%2E Plant Mol%2E Biol%2E Report%2E 20%3A 107%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Stangeland,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=An improved clearing method for GUS assay inArabidopsis endosperm and seeds&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=An improved clearing method for GUS assay inArabidopsis endosperm and seeds&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Stangeland,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Stingele%2C J%2E and Jentsch%2C S%2E %282015%29%2E DNA%2Dprotein crosslink repair%2E Nat Rev Mol Cell Biol 16%3A 455%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Stingele,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=DNA-protein crosslink repair&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=DNA-protein crosslink repair&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Stingele,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tian%2C G%2E%2C Lu%2C Q%2E%2C Zhang%2C L%2E%2C Kohalmi%2C S%2EE%2E%2C and Cui%2C Y%2E %282011%29%2E Detection of protein interactions in plant using a Gateway compatible Bimolecular Fluorescence Complementation %28BiFC%29 system%2E J Vis%2E Exp&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tian,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Detection of protein interactions in plant using a Gateway compatible Bimolecular Fluorescence Complementation (BiFC) system.&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Detection of protein interactions in plant using a Gateway compatible Bimolecular Fluorescence Complementation (BiFC) system.&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tian,&as_ylo=2011&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Torres%2DRosell%2C J%2E%2C Sunjevaric%2C I%2E%2C De Piccoli%2C G%2E%2C Sacher%2C M%2E%2C Eckert%2DBoulet%2C N%2E%2C Reid%2C R%2E%2C Jentsch%2C S%2E%2C Rothstein%2C R%2E%2C Aragon%2C L%2E%2C and Lisby%2C M%2E %282007%29%2E The Smc5%2DSmc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus%2E Nat Cell Biol 9%3A 923%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Torres-Rosell,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Torres-Rosell,&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tretyakova%2C N%2EY%2E%2C Groehler%2C A%2E%2C and Ji%2C S%2E %282015%29%2E DNA%2Dprotein cross%2Dlinks%3A Formation%2C structural identities%2C and biological outcomes%2E Acc Chem Res 48%3A 1631%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tretyakova,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=DNA-protein cross-links: Formation, structural identities, and biological outcomes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=DNA-protein cross-links: Formation, structural identities, and biological outcomes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tretyakova,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Tzafrir%2C I%2E%2C McElver%2C J%2EA%2E%2C Liu%2C C%2E%2C Yang%2C L%2EJ%2E%2C Wu%2C J%2EQ%2E%2C Martinez%2C A%2E%2C Patton%2C D%2EA%2E%2C and Meinke%2C D%2EW%2E %282002%29%2E Diversity of TITAN Functions in Arabidopsis Seed Development%2E Plant Physiol 128%3A 38%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Tzafrir,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Diversity of TITAN Functions in Arabidopsis Seed Development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Diversity of TITAN Functions in Arabidopsis Seed Development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Tzafrir,&as_ylo=2002&as_allsubj=all&hl=en&c2coff=1

Uhlmann, F. (2016). SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol 17: 399–412.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Vielle-Calzada, J.-P., Baskar, R., and Grossniklaus, U. (2000). Delayed activation of the paternal genome during seed development.Nature 404: 91.


Watanabe, K., Pacher, M., Dukowic, S., Schubert, V., Puchta, H., and Schubert, I. (2009). The STRUCTURAL MAINTENANCE OFCHROMOSOMES 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsisthaliana. Plant Cell 21: 2688–2699.


Wu, N. and Yu, H. (2012). The Smc complexes in DNA damage response. Cell Biosci 2: 5.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xu, P., Yuan, D., Liu, M., Li, C., Liu, Y., Zhang, S., Yao, N., and Yang, C. (2013). AtMMS21, an SMC5/6 complex subunit, is involved instem cell niche maintenance and DNA damage responses in Arabidopsis roots. Plant Physiol 161: 1755–1768.


Yan, S., Wang, W., Marqués, J., Mohan, R., Saleh, A., Durrant, W.E., Song, J., and Dong, X. (2013). Salicylic acid activates DNA damageresponses to potentiate plant immunity. Mol Cell 52: 602–610.


Yuan, D., Lai, J., Xu, P., Zhang, S., Zhang, J., Li, C., Wang, Y., Du, J., Liu, Y., and Yang, C. (2014). AtMMS21 regulates DNA damageresponse and homologous recombination repair in Arabidopsis. DNA Repair (Amst). 21: 140–147.


Zabrady, K., Adamus, M., Vondrova, L., Liao, C., Skoupilova, H., Novakova, M., Jurcisinova, L., Alt, A., Oliver, A.W., Lehmann, A.R., andPalecek, J.J. (2015). Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA. Nucleic AcidsRes 44: 1064–1079.


Zhang, S., Qi, Y., Liu, M., and Yang, C. (2013). SUMO E3 ligase AtMMS21 regulates drought tolerance in Arabidopsis thaliana. J IntegrPlant Biol 55: 83–95.


Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W., and Chua, N.-H. (2006). Agrobacterium-mediated transformation of Arabidopsis thalianausing the floral dip method. Nat. Protoc. 1: 641.


Zhao, X. and Blobel, G. (2005). A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomalorganization. Proc Natl Acad Sci U.S.A. 102: 4777–4782.


https://www.ncbi.nlm.nih.gov/pubmed?term=Uhlmann%2C F%2E %282016%29%2E SMC complexes%3A from DNA to chromosomes%2E Nat Rev Mol Cell Biol 17%3A 399%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Uhlmann,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=SMC complexes: from DNA to chromosomes&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SMC complexes: from DNA to chromosomes&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Uhlmann,&as_ylo=2016&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vielle%2DCalzada%2C J%2E%2DP%2E%2C Baskar%2C R%2E%2C and Grossniklaus%2C U%2E %282000%29%2E Delayed activation of the paternal genome during seed development%2E Nature&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Vielle-Calzada,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Delayed activation of the paternal genome during seed development&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Delayed activation of the paternal genome during seed development&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vielle-Calzada,&as_ylo=2000&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Watanabe%2C K%2E%2C Pacher%2C M%2E%2C Dukowic%2C S%2E%2C Schubert%2C V%2E%2C Puchta%2C H%2E%2C and Schubert%2C I%2E %282009%29%2E The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5%2F6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana%2E Plant Cell 21%3A 2688%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Watanabe,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Watanabe,&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wu%2C N%2E and Yu%2C H%2E %282012%29%2E The Smc complexes in DNA damage response%2E Cell Biosci&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Wu,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc complexes in DNA damage response&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=The Smc complexes in DNA damage response&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wu,&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xu%2C P%2E%2C Yuan%2C D%2E%2C Liu%2C M%2E%2C Li%2C C%2E%2C Liu%2C Y%2E%2C Zhang%2C S%2E%2C Yao%2C N%2E%2C and Yang%2C C%2E %282013%29%2E AtMMS21%2C an SMC5%2F6 complex subunit%2C is involved in stem cell niche maintenance and DNA damage responses in Arabidopsis roots%2E Plant Physiol 161%3A 1755%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Xu,&hl=en&c2coff=1


https://scholar.google.com/scholar?as_q=&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xu,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yan%2C S%2E%2C Wang%2C W%2E%2C Marqu�s%2C J%2E%2C Mohan%2C R%2E%2C Saleh%2C A%2E%2C Durrant%2C W%2EE%2E%2C Song%2C J%2E%2C and Dong%2C X%2E %282013%29%2E Salicylic acid activates DNA damage responses to potentiate plant immunity%2E Mol Cell 52%3A 602%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yan,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Salicylic acid activates DNA damage responses to potentiate plant immunity&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Salicylic acid activates DNA damage responses to potentiate plant immunity&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yan,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yuan%2C D%2E%2C Lai%2C J%2E%2C Xu%2C P%2E%2C Zhang%2C S%2E%2C Zhang%2C J%2E%2C Li%2C C%2E%2C Wang%2C Y%2E%2C Du%2C J%2E%2C Liu%2C Y%2E%2C and Yang%2C C%2E %282014%29%2E AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis%2E DNA Repair %28Amst%29%2E 21%3A 140%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Yuan,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yuan,&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zabrady%2C K%2E%2C Adamus%2C M%2E%2C Vondrova%2C L%2E%2C Liao%2C C%2E%2C Skoupilova%2C H%2E%2C Novakova%2C M%2E%2C Jurcisinova%2C L%2E%2C Alt%2C A%2E%2C Oliver%2C A%2EW%2E%2C Lehmann%2C A%2ER%2E%2C and Palecek%2C J%2EJ%2E %282015%29%2E Chromatin association of the SMC5%2F6 complex is dependent on binding of its NSE3 subunit to DNA%2E Nucleic Acids Res 44%3A 1064%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zabrady,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zabrady,&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang%2C S%2E%2C Qi%2C Y%2E%2C Liu%2C M%2E%2C and Yang%2C C%2E %282013%29%2E SUMO E3 ligase AtMMS21 regulates drought tolerance in Arabidopsis thaliana%2E J Integr Plant Biol 55%3A 83%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=SUMO E3 ligase AtMMS21 regulates drought tolerance in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=SUMO E3 ligase AtMMS21 regulates drought tolerance in Arabidopsis thaliana&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang,&as_ylo=2013&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhang%2C X%2E%2C Henriques%2C R%2E%2C Lin%2C S%2E%2DS%2E%2C Niu%2C Q%2E%2DW%2E%2C and Chua%2C N%2E%2DH%2E %282006%29%2E Agrobacterium%2Dmediated transformation of Arabidopsis thaliana using the floral dip method%2E Nat%2E Protoc&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhang,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhang,&as_ylo=2006&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Zhao%2C X%2E and Blobel%2C G%2E %282005%29%2E A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization%2E Proc Natl Acad Sci U%2ES%2EA%2E 102%3A 4777%26%23x&dopt=abstract

https://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Zhao,&hl=en&c2coff=1

https://scholar.google.com/scholar?as_q=A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Zhao,&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

DOI 10.1105/tpc.18.00043; originally published online April 29, 2019;Plant Cell

Gandha, Hana Jerábková, Sachihiro Matsunaga, Ueli Grossniklaus and Ales PecinkaMariana Diaz, Petra Pecinkova, Anna Nowicka, Celia Baroux, Takuya Sakamoto, Priscilla Yuliani

ArabidopsisSMC5/6 Complex Subunit NSE4A is Involved in DNA Damage Repair and Seed Development in

This information is current as of January 22, 2020

Supplemental Data /content/suppl/2019/05/18/tpc.18.00043.DC2.html /content/suppl/2019/04/29/tpc.18.00043.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

smc5/6 complex subunit nse4a is involved in dna damage ... · 144 and nse4b promoters revealed that...

Documents