rad51 localization and activation following dna damage

Published online 25 November 2003

RAD51 localization and activation followingDNA damage

Madalena Tarsounas, Adelina A. Davies and Stephen C. West*

Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK

The efficient repair of double-strand breaks in DNA is critical for the maintenance of genome stability.In response to ionizing radiation and other DNA-damaging agents, the RAD51 protein, which is essentialfor homologous recombination, relocalizes within the nucleus to form distinct foci that can be visualizedby microscopy and are thought to represent sites where repair reactions take place. The formation ofRAD51 foci in response to DNA damage is dependent upon BRCA2 and a series of proteins knownas the RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), indicating that thecomponents present within foci assemble in a carefully orchestrated and ordered manner. By contrast,RAD51 foci that form spontaneously as cells undergo DNA replication at S phase occur without the needfor BRCA2 or the RAD51 paralogues. It is known that BRCA2 interacts directly with RAD51 througha series of degenerative motifs known as the BRC repeats. These interactions modulate the ability ofRAD51 to bind DNA. Taken together, these observations indicate that BRCA2 plays a critical role incontrolling the actions of RAD51 at both the microscopic (focus formation) and molecular (DNAbinding) level.

Keywords: recombination; DNA repair; genomic instability; double-strand breaks; breast cancer

1. INTRODUCTION

When the structure of the DNA double helix was solvedin 1953, it immediately led Watson and Crick to suggesta mechanism for the replication of DNA (Watson & Crick1953). More difficult to imagine at that time was the wayin which a broken DNA helix could be repaired to main-tain the integrity of the genome. However, we now knowthat breakage of the sugar-phosphate backbone of DNAis a common event in cells that are irradiated or suffer theeffects of genotoxic agents, and that DBSs can be repairedby two primary pathways: HR and non-homologous end-joining (Kanaar et al. 1998). HR also provides a majorpathway by which stalled or broken replication forks canbe re-established, thereby allowing cell survival (Cox et al.2000; McGlynn & Lloyd 2003).

Simple organisms, such as bacteria, adapt for DNArepair and survival by initiating an ‘SOS response’ involv-ing the transcriptional de-repression of more than 20genes. One of these genes encodes RecA protein, whichis essential for recombination and recombinational repair.The induction of RecA is dramatic, with the levels of pro-tein increasing more than 15-fold within 30 min of theintroduction of DNA damage. Mammalian cells, however,do not elicit such a repair response, and, at most, there isonly a twofold transcriptional upregulation of proteinssuch as RAD51. Instead, in response to DNA damage,recombination proteins that are normally found diffusedthroughout the nucleus are rapidly relocalized and con-

* Author for correspondence ([email protected]).

One contribution of 18 to a Discussion Meeting Issue ‘Replicating andreshaping DNA: a celebration of the jubilee of the double helix’.

Phil. Trans. R. Soc. Lond. B (2004) 359, 87–93 87 2003 The Royal SocietyDOI 10.1098/rstb.2003.1368

centrated into sub-nuclear complexes that are microscopi-cally detected as foci. Thus, the overall effect is the same,in that the local protein concentration of key repairenzymes is increased as the cell prepares for repair.

Nuclear foci containing RAD51 form in response to avariety of DNA-damaging treatments (Haaf et al. 1995;Scully et al. 1997b). Recombination proteins that co-localize with RAD51 include RAD52 (Liu & Maizels2000; Lisby et al. 2001; Essers et al. 2002) and RAD54(Tan et al. 1999; Essers et al. 2002), the single-strandbinding protein RPA (Raderschall et al. 1999), and thetumour suppressors BRCA1 (Scully et al. 1997a) andBRCA2 (Chen et al. 1998a). The kinetics of localizationdiffer from one protein to another, indicating a sequentialassembly of one protein dependent upon another. Con-sistent with this interpretation, cell lines defective inBRCA2 fail to accumulate RAD51 foci after DNA-dam-aging treatments (Yuan et al. 1999; Godthelp et al. 2002;Tarsounas et al. 2003). The repair capacity of the cell istherefore compromised.

RAD51 foci are also found in undamaged S-phase cells,where they are thought to identify sites where stalled orbroken replication forks undergo repair (Tashiro et al.1996; Raderschall et al. 1999). The S-phase and damage-induced foci appear to be distinct from each other,because BRCA2 is not required for the formation ofRAD51 foci in non-irradiated S-phase cells (Tarsounas etal. 2003).

Mutations in either of the breast-cancer-susceptibilitygenes BRCA1 or BRCA2 are associated with a predis-position to breast and ovarian cancers (Welcsh et al.2000). The products of these genes, the BRCA1 andBRCA2 proteins, are required for the recombinationalrepair of DSBs (Moynahan et al. 1999, 2001; Xia et al.

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88 M. Tarsounas and others RAD51 and DNA repair

3418 amino acidsNLS

RAD51 binding

BRC repeatsOBhelical domain

DSS1 binding

ssDNA bindingBRAF35 binding

P/CAF binding

1 2 3

1863 amino acids

M/R/N binding

BRCT repeats

BRCA2BACH1CtIPRNA pol II

BARD1 binding

RING finger NLS(a)

(b)

Figure 1. Functional domains of (a) BRCA1 and (b) BRCA2. Both proteins are large polypeptides (1863 and 3418 aminoacids, respectively) that interact with each other and with a number of other proteins. For example, BRCA1 interacts with theBARD1 (BRCA1-associated RING finger domain) protein, which is required for ubiquitin ligase activity, theMRE11/RAD50/NBS1 (M/R/N) recombination/repair complex, a putative DNA helicase BACH1, the CtBp-interactingprotein CtIP and RNA polymerase II. The sites of the RING finger, the nuclear-localization signals (NLS) and the BRCTdomains are indicated. Similarly, BRCA2 interacts with the histone acetylase P/CAF, the 35 kDa BRCA2-associated factorBRAF35 and DSS1 (a protein deleted in split hand/split foot syndrome). The sites of the eight BRC repeats, six of whichinteract with RAD51, the oligonucleotide binding (OB) domains that interact with single-stranded DNA, and the carboxy-terminal NLS are indicated.

2001), and have been shown to associate with each otherand with RAD51 (Mizuta et al. 1997; Scully et al. 1997a,b;Chen et al. 1998a; Marmorstein et al. 1998; Yuan et al.1998). Consequently, a primary outcome of BRCA1 andBRCA2 deficiency is chromosomal instability due to aninability to repair DNA lesions by HR (Patel et al. 1998;Tutt et al. 1999; Yu et al. 2000).

BRCA1 (208 kDa) and BRCA2 (384 kDa) are largeproteins that exhibit direct interactions with several otherproteins, many of which are required for efficient DNArepair (figure 1). Cell lines defective in BRCA1 or BRCA2show gross chromosomal rearrangements, and there areindications of chromosome breakage (Xu et al. 1999; Yuet al. 2000). Similarly, cells derived from tumours takenfrom individuals predisposed to cancer through BRCA1and BRCA2 germline mutations show evidence of genomeinstability. The interaction of BRCA2 with RAD51,together with the recombination/repair-defective pheno-type of BRCA1- or BRCA2-defective cell lines, indicatesthat the main defect leading to genome instability in thesecells lies in RAD51-mediated DNA repair systems. Inter-actions that occur between the BRCA proteins andRAD51, and the influence that these tumour suppressorsexert over RAD51 activity, are therefore of central impor-tance to our understanding of mechanisms of genomicinstability and tumorigenesis.

2. RESULTS AND DISCUSSION

(a) Formation of repair foci in response to IRWhen an asynchronous culture of HeLa cells was ana-

lysed for the presence of RAD51 foci using immunofluo-rescence staining, ca. 10–15% of the cells were found tocontain RAD51 foci (figure 2a(i)). These are thought torepresent the population of cells in the culture that areundergoing S phase and are actively replicating their DNA

Phil. Trans. R. Soc. Lond. B (2004)

(Tarsounas et al. 2003). Following treatment with 10 Gyof IR, however, the number of cells containing RAD51foci increases to more than 50% (figure 2a(ii)). When IR-induced foci were analysed for the presence of BRCA2,good co-localization with RAD51 was observed (figure2b). Similarly, RAD51 co-localizes with RAD54 (figure2c(i)) and shows partial localization with RPA (figure2c(ii)). The accumulation of recombination proteins suchas RAD51 and RAD54 in IR-induced foci supports thenotion that foci represent sites where repair reactions takeplace. Moreover, the presence of the single-strandedDNA-binding protein RPA indicates the presence of DNAintermediates that are undergoing repair or replication.

(b) Cell lines defective in the RAD51 paraloguesform replication-associated but not IR-induced

RAD51 fociPreviously, we showed that cells carrying a truncating

mutation in BRCA2 failed to form IR-induced RAD51foci yet exhibited S-phase foci as normal, leading us tosuggest that S-phase and IR-induced RAD51 fociassemble by distinct pathways with defined proteinrequirements (Tarsounas et al. 2003). Cells defective inany of the five RAD51 paralogues (RAD51B, RAD51C,RAD51D, XRCC2 and XRCC3), which are required fornormal levels of HR and resistance to IR (Thacker 1999),also fail to form RAD51 foci in response to IR, as shownin figure 3 and elsewhere (Bishop et al. 1998; Takata etal. 2000, 2001; O’Regan et al. 2001). However, weobserved that replication-associated RAD51 foci werepresent in ca. 9–13% of the total cell population in asyn-chronous irs1 (XRCC2�) and irs1SF (XRCC3�) cultures.A similar percentage of the wild-type control V79 cellsexhibited RAD51 foci. In all three non-irradiated celllines, RPA showed good co-localization with RAD51(figure 4). We conclude that, like BRCA2, the requirement

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RAD51 and DNA repair M. Tarsounas and others 89

α-BRCA2

α-RAD54

α-RAD51

α-RPA

α-RAD51

α-RAD51 merge

(a)

(b)

(c)

(i) (ii)

Figure 2. Co-localization of recombination/repair proteins in human cells following IR. (a) HeLa cells were irradiated with10 Gy IR and allowed to recover for 3 h. (i) Non-irradiated and (ii) irradiated cultures were stained with an anti-RAD51polyclonal antibody FBE1 as described (Tarsounas et al. 2003). (b) BRCA2 (green) and RAD51 (red) foci were visualized 1 hafter IR using the rabbit affinity-purified anti-BRCA2 pAb PC146 (Oncogene) diluted 1/1000 and mouse anti-RAD51 mAb14B4 (AbCam) diluted 1/200. (c) RAD54 (green) and RAD51 (red) foci were visualized 4 h after IR using rabbit anti-RAD54pAb SWE27 diluted 1/1000 and mouse anti-RAD51 mAb 14B4 (AbCam) diluted 1/200. RPA (green) and RAD51 (red) fociwere visualized 4 h after IR using mouse anti-RPA 70 kDa subunit mAb 70A (a gift from Dr R. Wood) diluted 1/100 andrabbit anti-RAD51 pAb FBE1 diluted 1/500. Protein co-localization is indicated by the yellow foci in the merged images.

for the RAD51 paralogues is by-passed in the context ofS-phase replication-associated RAD51 foci. BRCA2 andthe RAD51 paralogues are, however, necessary for theproper assembly of repair foci following the introductionof de novo DNA damage, and are likely to play a directrole in DNA repair or the regulation of protein activitiesrequired for efficient repair reactions.


(c) Interactions between BRCA2 with RAD51BRCA2 contains a series of eight degenerate motifs

(Bork et al. 1996), six of which have been shown to bindRAD51 (Wong et al. 1997; Chen et al. 1998b). TheseBRC motifs are approximately 30 amino acids long, andare interspersed along a 1200-amino-acid central regionof the protein (figure 1). There is an additional unrelated

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V79

0 Gy

10 Gy

irs1 irs1SF

Figure 3. RAD51 focus formation in XRCC2- and XRCC3-defective cell lines. irs1 (XRCC2-deficient) and irs1SF (XRCC3-deficient) hamster cell lines, and the control line V79, were analysed for RAD51 foci using the rabbit pAb FBE2 at 1/500dilution, with or without irradiation (10 Gy, 3 h recovery).

α-RAD51

α-RPA

merge

V79 irs1 irs1SF

Figure 4. Co-localization of RAD51 and RPA in non-irradiated XRCC2- and XRCC3-defective cells. RAD51 and RPA werevisualized at replication-associated foci using anti-RAD51 pAb FBE2 and the anti-RPA mAb 70A, respectively. Co-localizationof RAD51 (red) and RPA (green) is indicated by the yellow foci in the merged images.

RAD51-binding site located at the carboxyl terminus ofBRCA2. Over-expression of a single BRC repeat in rodentcells is sufficient to cause decreased HR, radiation hyper-sensitivity, and a loss of G2/M checkpoint control, pre-sumably because it acts in a dominant negative mannerby sequestering RAD51 (Chen et al. 1999a; Stark et al.2002).


To determine the consequences of interactions betweenBRC motifs and RAD51, we previously analysed theeffects of synthetic peptides corresponding to the BRCrepeats on the DNA-binding properties of RAD51 (Davieset al. 2001). We found that interactions between RAD51and BRC3 or BRC4 inhibited the DNA-binding proper-ties of RAD51. As shown in figure 5a (compare lanes b

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– DNA

BRC4– +

– RAD51–DNAcomplex

P

RAD51– + + +

a b c d

(a) (b)

RAD51

RAD51+

BRC4

RAD51+

BRC4-P

–

Figure 5. Interactions between RAD51 and the BRC4 region of BRCA2. (a) Purified RAD51 protein (4 �M) in 20 mM Tris–HCl pH 7.5, 1 mM EDTA, 0.5 mM DTT, 0.2 M KOAc and 10% glycerol, was incubated for 15 min at 37 °C either alone(lane b), with the BRC4 peptide (lane c) or with phosphorylated BRC4 (lane d). Reactions were then supplemented withbinding buffer (50 mM triethanolamine–HCl pH 7.5, 0.5 mM Mg(OAc)2, 2 mM ATP, 1 mM DTT and 100 �g ml�1 BSA)and 32P-5�-end-labelled tailed linear duplex DNA (5 �M) essentially as described (Davies et al. 2001). All peptides werepresent at 24 �M. Protein–DNA complexes were fixed with glutaraldehyde and analysed by 0.8% agarose gel electrophoresisfollowed by autoradiography. (b) Electron microscopic visualization of nucleoprotein filaments formed by RAD51 in thepresence or absence of BRC4 peptide as indicated. The amino acid sequence of the 69-amino-acid peptide corresponding tothe BRC4 repeat (amino acids 1511–1579) of BRCA2 is ERDEKIKEPTLLGFHTASGKKVKIAKESLDKVKNLFDEKEQGTSEITSFSHQWAKTLKYREACKDLELA. The phosphorylation site (Thr1526) is underlined.

and c) and figure 5b, a 69-amino-acid long BRC4 peptide,blocked nucleoprotein filament formation by RAD51, asdetermined by band-shift assays and electron microscopy.Since recombination reactions take place within nucleo-protein filaments (as shown in figure 5b), interactionsbetween RAD51 and the BRC4 peptide would lead toinactivation of RAD51. Single amino acid changes withinthe conserved BRC motifs were found to eliminate thisinhibitory effect, such that RAD51 bound DNA normallyin the presence of mutant peptides (Davies et al. 2001).It is reasonable to think that these peptide studies give ussome insight into the interactions that occur with full-sizeBRCA2, and that a novel negative control mechanism ofBRCA2 over RAD51 has been uncovered.

The role of BRCA2 in recombinational repair, however,cannot simply be one of negative control. As describedabove, BRCA2 is required for the accumulation ofRAD51 at damage-induced foci, so it is plausible thatBRCA2 provides a scaffold that keeps RAD51 inactiveuntil the moment when DNA damage occurs. At thattime, BRCA2 and RAD51 relocalize to repair foci, wherethe RAD51 needs to be released for nucleoprotein fila-ment formation.


The BRC4 region of BRCA2 interacts with RAD51such that it prevents RAD51 monomer–monomer interac-tions (Davies et al. 2001; Pellegrini et al. 2002), whichin turn blocks filament formation on DNA. The crystalstructure of the RAD51–BRC4 complex indicates that 28amino acids of BRC4 are in close contact with RAD51,with the highly conserved residues 1524FHTASGK1530being part of a hairpin loop that is involved in criticalhydrophobic and polar interactions with RAD51(Pellegrini et al. 2002). Thr1526 is essential for confor-mational stability of the loop, and mutations at equivalentpositions in BRC1, BRC2 and BRC7 of BRCA2 havebeen reported in individuals with breast cancer.

Because it is possible that modification of this aminoacid ‘controls’ BRC–RAD51 interactions, we investigatedthe effect of phosphorylation of BRC4 at this site by syn-thesizing a 69-amino-acid BRC4 peptide phosphorylatedat Thr1526. As shown in figure 5a (lane d) and figure 5b,phosphorylation of Thr1526 blocked the ability of BRC4to interact with RAD51, and thus RAD51 bound DNAand formed nucleoprotein filaments normally. It is knownthat BRCA1 (Scully et al. 1997b; Cortez et al. 1999) andRAD51 (Yuan et al. 1998; Chen et al. 1999b; Saintigny

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et al. 2001) are phosphorylated in response to IR; thus itis possible that modification events might play a role inthe release of RAD51 from BRCA2. Our results show thatinteractions between BRC motifs and RAD51 are highlysensitive to even the most subtle changes, and it is attract-ive to speculate that damage-dependent modifications toBRCA2 at the BRC motifs could result in significantchanges to the activation state of RAD51, possibly byallowing the release of RAD51 for nucleoprotein filamentformation at sites of repair.

The authors thank the members of the West laboratory forsuggestions, and the CR-UK cell production and peptide syn-thesis units for their help. Cancer Research UK and the SwissBridge Foundation supported this work. Madalena Tarsounaswas supported by a post-doctoral fellowship from the EuropeanMolecular Biology Organisation. The authors thank AndrzejStasiak for carrying out electron microscopic analyses of BRC-RAD51 interactions and providing the images shown in figure 5.

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GLOSSARY

DSB: double-strand breakHR: homologous recombinationIR: ionizing radiation

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rad51 localization and activation following dna damage

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