photorepair mutants of arabidopsis

Photorepair Mutants of ArabidopsisAuthor(s): C.-Z. Jiang, J. Yee, D. L. Mitchell and A. B. BrittSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 94, No. 14 (Jul. 8, 1997), pp. 7441-7445Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/42697 .

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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7441-7445, July 1997 Genetics

Photorepair mutants ofArabidoj C.-Z. JIANG*, J. YEE*, D. L. MITCHELL?, AND A. B. BRIT

*Section of Plant Biology, University of California, Davis, CA 95616; and tDep Center, Smithville, TX 78957

Communicated by Richard B. Setlow, Brookhaven National Laboratory,

ABSTRACT UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

The biological effects of UV light have been extensively studied in microbes and mammals, where UV irradiation has been shown to have both toxic and mutagenic effects. The two primary UV-induced DNA damage products are the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6-4) pyrimidinone dimer (the 6-4 product), with 6-4 products mak- ing up an estimated 10-25% of all dimers (1). Pyrimidine dimers are known to inhibit microbial and mammalian DNA replication; they have been shown to act in cis to directly inhibit the progress of DNA polymerase, as well as in trans to inhibit the initiation of replication in both microbes and mammals (2, 3). Dimers are, under some circumstances, bypassed in an error-prone fashion, and this trans-lesion synthesis can result in the induction of mutations (4-8). RNA polymerase has also been shown to "stall" at both CPDs and 6-4 photoproducts (9-12); in the absence of repair, a single pyrimidine dimer may be sufficient to eliminate expression of a transcriptional unit. Because every pyrimidine dimer acts as a block to transcription and replication, any living tissue, even one in which cell division does not occur, must either avoid or repair UV- induced DNA damage if it is to survive.

The CPD and the 6-4 product have very different structures (Fig. 1) and, therefore, different capacities to form both accurate and inaccurate base pairs (13-15). In recent years the relative contributions of these two lesions to UV-induced cytotoxicity and mutagenicity has been the subject of debate and has been addressed in human and rodent cell lines through a combination of shuttle-vector studies and investigations into the effects of various repair deficiencies. Studies using shuttle vectors may not directly reflect the effects of persisting

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. ? 1997 by The National Academy of Sciences 0027-8424/97/947441-5$2.00/0 PNAS is available online at http://www.pnas.org.

vsis rT*t

artment of Carcinogenesis, University of Texas M. D. Anderson Cancer Research

Upton, NY, May 7, 1997 (received for review December 9, 1996)

genomic lesions, because plasmid DNAs lack many of the qualities of genomic DNA, such as higher-order chromatin packing or in some cases the presence of an actively transcribed region. Mammalian repair deficient mutants display a complex phenotype, simultaneously affecting both CPD and 6-4 product removal, with differential effects on global vs. transcription-coupled repair (16); interpretation of results obtained with these mutants is not straightforward. Ideally, one would like to induce just CPDs, or just 6-4 products, in genomic DNA to determine the relative effects of these very different types of pyrimidine dimers on cell growth, transcription, DNA replication, cell cycle arrest, apoptosis, and mutagenesis. Herein we demonstrate that it may be possible, by using Arabidopsis mutants, to generate living plants with a nuclear genome containing only one or the other type of dimer.

MATERIALS AND METHODS

Plant Material. Isolation of Arabidopsis thaliana strain Landsberg erecta, transparent testa 5 (tt5) UVresistance I (uvrl) has been described (17). The uvrl line used in these experiments had been backcrossed three times to its tt5 progenitor. The newly isolated photoreactivation mutants were also backcrossed once to their uvrl tt5 progenitor. Arabidopsis seedlings were grown at 22?C with continuous illumination under cool white lamps (photosynthetically active radiation = 100 pmol per m2 per sec) filtered through 0.005 ml Mylar (Golden State Plastics, Sacramento, CA). This filter effectively eliminates wavelengths of less than 310 nm (50% cut off at 320 nm), transmitting less than 5% of the minor peak at 313 nm emitted by the cool white lamps and greater than 80% of the next emission peak at 365 nm.

Isolation of Photoreactivation Mutants. Seeds of uvrl tt5 were mutagenized with 0.15% ethylmethane sulfonate. These M1 seeds were sown and allowed to self-pollinate in the absence of UV radiation (wavelengths <400 nm) under UV3 Plexiglas (Golden State Plastics, Sacramento, CA). The resulting M2 seeds were collected in 14 separate batches, and approximately 1,000 2-week-old M2 plants from each batch were screened for UV sensitivity. This screen was performed with a germicidal lamp at a UVC dose of 200 J/m2 applied over 11 sec. After UV irradiation, seedlings were grown under Mylar-filtered cool white lamps for a week. Plants that exhib- ited a stress response to the challenge UV dose (scored as browning, puckering, and rolling of the leaf edges) were tagged as putative UV-sensitive mutants. The putative mutants were allowed to self-pollinate, and seeds were collected from indi- vidual plants. Each mutant family was rescreened to confirm the heritability of the mutant phenotype.

Complementation Tests. UV-sensitive mutant lines were backcrossed to their uvrl tt5 progenitor to produce an F1 generation. FA individuals were self-pollinated and harvested individually, and their progeny were scored for UV sensitivity as described above. All mutants segregated 3:1 for UV resis-

Abbreviation: CPD, cyclobutane pyrimidine dimer. tTo whom reprint requests should be addressed. e-mail: abbritt@ ucdavis.edu.

7441



7442 Genetics: Jiang et al.

O O

H H H3

ii

H 'N ^ C C 3

||NH2

1 CH3N.*

pyrimidine (6-4) pyrimidinone TpC dimer FIG. 1. Structures of the cis-syn cyclobutane thymine dimer (a

CPD) and the T.C pyrimidine (6-4)pyrimidinone dimer (a 6-4 product).

tance vs. sensitivity in the F2 backcross generation, indicating that the mutations were recessive. The UV-sensitive backcrossed mutant lines were crossed to each other, and approximately 50 F1 hybrid individuals from each cross were scored for UV sensitivity. F1 families with 100% UV-sensitive plants were scored as the product of a cross between two plants in the same complementation group; families with 100% UV- resistant progeny were scored as the product of a cross between plants carrying mutations in different complementation groups.

Assay of Pyrimidine Dimers. Seeds were sterilized in 0.525% sodium hypochlorite/0.1% Triton X-100 for 10 min and, after five washes with sterilized water, germinated on vertically oriented nutritive agar plates. Five-day-old seedlings were irradiated with a UVB dose of 1.4 kJ/m2, at a dose rate of 3.3 mW/cm2, for 45 sec and harvested at different times ranging from 0 to 4 h after UV irradiation. Recovery was under Mylar-filtered cool white lamps. The UVB source was a UV transilluminator (Fisher) with a peak output at 305 nm, filtered through a 0.005-ml cellulose acetate sheet (Golden State Plastics, Sacramento, CA) that absorbs the UVC (<280 nm) fraction of the emitted light. UVB intensity was measured with a UVB radiometer with a peak response at 310 nm, and a 50% response at 280 and 340 nm (Ultraviolet Products, San Gabriel, CA). DNA from 5-day-old seedlings was isolated by the cetyltrimethyl ammonium bromide method (18). DNA con- centration was measured by the fluorometric method (19) with a Hoefer DyNA fluorometer. CPDs and 6-4 products were quantified by a lesion-specific radioimmunoassay (20). Four micrograms of DNA was required for each assay of 6-4 products; one microgram of DNA was used for each assay of CPDs.

Root Elongation Assay for UV Sensitivity. Prior to UV irradiation, seedlings were grown on vertically oriented agar plates for 3 days at 22?C in the presence of light. After UVB irradiation, the plates were rotated by 90? and returned to the 22?C growth room for another 3-day period. At the end of this time, additional root growth, measured as root elongation at a 90? angle to the growth prior to irradiation, was quantified by using an eyepiece micrometer.

Proc. Natl. Acad. Sci. USA 94 (1997)

RESULTS AND DISCUSSION

Isolation of Mutants Defective in Photoreactivation. Pho- tolyases are specialized repair proteins that bind to dimers and, upon absorption of a photon of the appropriate wavelength (350-450 nm), directly reverse the damage in an error-free manner. CPD-specific photolyases have been detected in and cloned from a broad range of species and are classified, on the basis of their sequence similarities, as type I or type II. Type I photolyases are found in a variety of bacteria and lower eukaryotes, and type II enzymes, also termed "metazoan" photolyases, are found in multicellular animals including in- sects, fish, and marsupials (21, 22).

Recently, a 6-4-product-specific photolyase activity was discovered in extracts from Drosophila melanogaster cells (23, 24) and more recently in extracts from two vertebrates: Crotalus atrox (rattlesnake) and Xenopus laevis (South African clawed toad) (25). The sequence of the gene encoding this activity most closely resembles a type I CPD photolyase (26), although the enzyme is strictly specific for 6-4 products. Arabidopsis seedlings have been shown to rapidly eliminate both CPDs and 6-4 products from the nuclear genome via a visible-light-dependent pathway (27, 28). A light-dependent pathway for the removal of 6-4 products has also been demonstrated in cucumber (29). The 6-4 photoreactivating activity is constitutively expressed inArabidopsis seedlings, and the CPD-specific activity requires prior exposure of the seedlings to visible light for optimal expression (28). This differential regulation suggests that the activities are encoded by two different genes.

To determine the number of genes involved in photoreactivation of UV-induced DNA damage in Arabidopsis, we isolated a number of photorepair-defective mutants. Arabi- dopsis seedlings can eliminate nuclear 6-4 products from the nuclear genome in the absence of photoreactivating light, albeit inefficiently. To eliminate this "background" repair activity and thus enhance the UV-sensitive phenotype of any potential 6-4 photoreactivation mutants, we screened for secondary mutations enhancing the UV sensitivity of a uvrl mutant line defective in the light-independent repair of 6-4 products (17). Approximately 14,000 mutagenized uvrl plants were grown under UV-free conditions and allowed to self- pollinate. The resulting M2 seeds were collected in 14 separate batches, and approximately 1,000 M2 plants from each batch were screened for UV sensitivity, detected as a leaf-rolling response to low doses of UVC. A total of six mutants displaying a visible-light-dependent UV-sensitive phenotype were confirmed. These mutations fell into only two complementation groups. The first group of mutants, represented by at least three independently derived isolates, was assigned the designation uvr2, and the second complementation group, represented by a single isolate, was assigned the designation uvr3.

The rate of repair of 6-4 products and CPDs was deter- mined in the mutants and contrasted with that of their uvrl progenitor. The uvr2 mutant was found to be deficient in the photoreactivation of CPDs, but proficient in the photoreactivation of 6-4 products (Fig. 2 A and B). Conversely, the uvr3 mutant was found to be deficient in the photoreactivation of 6-4 products but proficient in the repair of CPDs (Fig. 2 C and D). These results indicate that the 6-4 and CPD photolyases are encoded by two distinct genes.

The uvr2 Mutation Is Genetically Linked to a Type II Photolyase Sequence. The CPD photolyase deficient mutant (uvr2) was then used to determine which of the several photolyase-like sequences in the Arabidopsis genome corre- sponded to our mutant locus. Arabidopsis encodes at least two expressed genes, HY4 (30) and PHH1 (31), with sequence similarity to the type I photolyase. However, neither of these genes encode a photolyase activity on expression in Escherichia coli (32, 33), and HY4 is known to encode a blue light



Genetics: Jiang et al.

A: Photorepair of CPDs

100

4l

o 1

0 1 2 3 4 5 00 time (hours)

B: Photorepair of 6-4s

0 2 3 4 5

I Photoreair of 6-4 s

by uvrl vs. uvrl uvr3. O, uvrl; A, uvrl uvr2; O, uvrl uvr3.

photoreceptor (30). Degenerate PCR primers previously used to amplify type II CPD photolyase genes from a variety of organisms (22) were used to amplify a related sequence from an Arabidopsis cDNA library (34). The resulting 324-bp PCR product was then used as a probe to isolate a larger 1,080-bp clone from the same library. This partial cDNA, which lacks a translation start site, displays remarkable sequence identity at

I 0 ?

yases, including a 67% identity, and 83% similarity, in a contiguous stretch of 196 amino acids (from amino acids 267

to 462 of the opossum Monodphus domstica sequence). The entrcN sqeeotie CPD photolyase homolog

FIG. 2. uenBan accession no. X99301) has rdefective in theen photoractivationshed

(35).

two from each of two independent experiments. Error bars are I Photoreactivation of 6-4 products by uvrl vs. uvrl uvr2. (C) Photoreact by uvrl vs. uvrl uvr3. 0, uvrl'; A, uvrl uvr2; w, uvrl uvr3.

Usingphotoreeptor (30). Degenerate PCR primers reiously used to amplify type II CPD photolyase gene, we amplified and sequenced the 2.5-kb

genomic DNA from both our ttS uvrl progenitor strain and its

organisms (22) were used to amplify a related sequence from an Arabidopsis cDNA library (34). The resulting 324-bp PCR product was then used as a probe to isolate a larger 1,080-bp clone from the same library. This partial aDNA, which lacks a translation start site, displays remarkable sequence identity at the amino acid leve l to previously cloned me tazoan photol-

yases, including a 67% identity, and 83% similarity, in a contiguous stretch of 196 amino acids (from amino acids 267 to 462 of the opossum Monodelphus domestica sequence). The

entire wDNA sequence of this CPD photolyase homolog (GenBank accession no. X99301) has recently been published (35).

U sing PCR primers bordering t he transcribed region of this type II photolyase gene, we amplified and sequenced the 2.5-kb genomic DNA from both our tt5 uvrl progenitor strain and its uvr2 derivative. The uvr2 line was found to carry a C -- T transition in the 259th nucleotide (using the adenosine of the first AUG as nucleotide 1). This results in a nonsense mutation in the first exon of the gene.

To determine whether the mutant type II photolyase sequence was genetically linked to the CPD photolyase deficiency, a restriction fragment length polymorphism between two Arabidopsis ecotypes Landsberg erecta (Ler, the genetic background in which the uvr2 mutation was isolated) and Columbia (Col, a strain proficient in CPD photoreactivation)

Proc. Natl. Acad. Sci. USA 94 (1997) 7443

C: Photorepair of CPDs

100 I

0

c. -

I 0 0 1 2 3 4 5

time (hours)

D: Photorepair of 6-4s

100 C . - .

4)

.I

1 0 1 0 1 2 3 4 5

of pyrimidine dimers. Each data point represents the average of four points, SD. (A) Photoreactivation of CPDs by uvrl vs. uvrl uvr2 mutants. (B) ivation of CPDs by uvrl vs. uvrl uvr3. (D) Photoreactivation of 6-4 products

was identified on Southern blots probed with the type II photolyase sequence. This polymorphism enabled us to track the segregation of the Ler vs. Col alleles of the type II photolyase homolog.

The uvrl uvr2 tt5 Ler line was crossed to the wild-type Col line and F2 segregants were classified in terms of their UV sensitivity (assayed as a leaf-rolling response to UVC at 200 J/m2). Of 109 F2 plants screened, 25 were tentatively identified as more UV sensitive than their uvrl tt5 progenitor in the presence of photoreactivating light. These presumptive uvr2 homozygotes were self-pollinated and their F3 progeny were further characterized in terms of their UV sensitivity. This screen confirmed that 20 F2 families were homozygous for a visible-light-dependent UV-sensitive phenotype.

DNA was prepared from seedlings from each of these UV-sensitive families and characterized as to the presence or absence of the Ler restriction fragment length polymorphism. All 20 families were homozygous for the Ler polymorphism at the type II sequence, and UV-resistant or segregating families were homozygous or heterozygous for the Col restriction fragment length polymorphism (data not shown). The genetic linkage between the mutant DNA sequence and the repair- deficient phenotype strongly suggests that the Arabidopsis CPD photolyase is encoded by a type II (metazoan) photolyase. However, there is a remote possibility that this type II photolyase homolog encodes, instead, a light receptor that regulates the expression of the actual photolyase gene.



7444 Genetics: Jiang et al.

An Arabidopsis mutant defective in CPD photoreactivation has recently been reported (36). This mutant maps to the same position as our uvr2 mutation, is known to carry a nonsense mutation in the same gene (37), and is also termed uvr2.

The Two Classes of Photoreactivation Mutants Differ in Their Sensitivity to UV. The biological effect of any persisting DNA damage product will depend on the nature of its interaction with both DNA and RNA polymerases. The ability of a polymerase to bypass UV-induced damage depends on the type of polymerase involved and may also vary from species to species. The structures of the 6-4 product and the CPD are very different, and the two lesions may have different toxic and mutagenic properties. Double mutants defective in both exci- sion repair (uvrl) and either the CPD photolyase (uvr2) or the 6-4 photolyase (uvr3) are unable to eliminate these DNA damage products from the nuclear genome. Thus the uvr mutants provide us with the opportunity to determine the severity of the toxic effects of nuclear CPDs (in the absence of 6-4 products) and nuclear 6-4 products (in the absence of CPDs). It should be noted, however, that the uvr2 mutation does not affect repair of CPDs in the plastid or mitochondrial genomes; these lesions are not repaired in wild-type Arabi- dopsis seedlings (38). A similar, although preliminary, result suggesting a lack of repair of 6-4 products has been obtained for the spinach plastid genome.?

To determine the relative toxicity of nuclear CPDs vs. nuclear 6-4 products in Arabidopsis, 5-day-old seedlings were irradiated with UVB and the resulting effects on growth were assayed by measuring root growth during a 3-day period after irradiation. The inhibitory effects of UV on root growth were photoreactivatable (Fig. 3), and mutants deficient in photorepair displayed a deficiency in photoreactivation of root growth. In the presence of photoreactivating light, mutants defective in CPD photolyase were approximately 7-fold more sensitive to UVB than their uvrl progenitor line, when sensitivity was measured as the UVB dose required to inhibit growth to l/e (37%) of the unirradiated value. All three independently derived uvr2 isolates showed the same degree of UV sensitivity (data not shown). The uvrl uvr2 mutants lacked any detectable photoreactivation of root growth; mutants defective in photoreactivation of CPDs were equally sensitive to UVB in the presence or absence of visible light.

In contrast, the mutant defective in 6-4 photorepair was more sensitive to UVB than its uvrl progenitor line, but less sensitive than the CPD photolyase mutant. The uvrl uvr3 double mutant was approximately 3-fold more UV-sensitive than its uvrl progenitor. This suggests that although the persistence of 6-4 products does have a significant growth- inhibitory effect, most of the growth-inhibitory effect of UVB is due to the induction of CPDs. This result does not indicate, however, that the 6-4 product is a less toxic lesion than the CPD. To determine the relative toxicity of these two classes of lesions, we need to determine the relative rate of induction of 6-4 products vs. CPDs in our experimental system, and we have not yet obtained this value. However, typical in vivo ratios for the induction of CPDs vs. 6-4 products range from 3:1 to 9:1 (1, 39), suggesting that the average 6-4 photoproduct, in comparison to the average CPD, has an equivalent or greater inhibitory effect on the elongation of theArabidopsis seedling's root.

It should be noted, however, that other classes of DNA damage, including oxidative damage, crosslinks, and Dewar isomers of 6-4 products, might be induced at a significant rate by our UVB treatment, and our experiments do not address the possible contributions of these lesions to the inhibitory effects of UVB on seedling growth.

?Hada, M., Kameoka, S., Hashimoto, T. & Shin, M., 12th International Congress on Photobiology, Sept. 1-6, 1996, Vienna, Austria.

Proc. Natl. Acad. Sci. USA 94 (1997)

A: Recovery in the dark

c 100 0

0 1 2 3 4 5 6 7 0

UV-B dose, kJ/m2

B: Recovery in the light

100?

5 ^13

0)

0

o 1 I I I i I I

0 1 2 3 4 5 6 7 UV-B dose, kJ/m2

FIG. 3. Effect of UV radiation on the growth of the photoreactivation mutants and their uvrl progenitor. Values represent the average of measurements of root growth on 20 seedlings 3 days after irradiation; error bars are 1 SD. o, uvrl, A, uvrl uvr2, a, uvrl uvr3.

The biological consequence of any particular environmental DNA damaging agent is a complex function of the frequency of induction of DNA damage products; the immediate effects of each lesion on transcription, replication, and cell cycle control; and the rate at which each type of lesion is repaired. By using mutants defective in the repair of CPDs or 6-4 products, we have shown that both classes of pyrimidine dimers play a significant role in the inhibition of root elongation by UVB. The specific effects of each class of pyrimidine dimers on DNA replication, cell cycle arrest, transcription, and mutagenesis remain uncertain. The uvr2 and uvr3 mutants of Arabidopsis provide us with a valuable tool to further dissect the mechanistic basis of UV-induced genotoxicity.

This work was supported by a grant from the United States Department of Agriculture National Competitive Research Initiative Grants Program in Plant Genetic Mechanisms, Agreement 94-37301- 0564.

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photorepair mutants of arabidopsis

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