Role of BRCA2 in Control of the RAD51 Recombination and DNA Repair Protein

Molecular Cell, Vol. 7, 273–282, February, 2001, Copyright 2001 by Cell Press

Role of BRCA2 in Control of the RAD51 Recombination and DNA Repair Protein Adelina A. Davies,* Jean-Yves Masson,* Michael J. McIlwraith,* Alicja Z. Stasiak,† Andrzej Stasiak,† Ashok R. Venkitaraman,‡ and Stephen C. West*§ * Imperial Cancer Research Fund Clare Hall Laboratories South Mimms, Hertfordshire EN6 3LD United Kingdom † Laboratoire d’Analyse Structurale Universite´ de Lausanne 1015 Lausanne Switzerland ‡ University of Cambridge CRC Department of Oncology Wellcome Trust Centre for Molecular Mechanisms of Disease Cambridge Institute for Medical Research Cambridge CB2 2XY United Kingdom

Summary Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis. Introduction Approximately 5% of breast cancers result from a genetic predisposition to the disease caused by a germline mutation in BRCA1 or BRCA2. Mutations in these breast cancer susceptibility genes are also often associated with an increased risk of ovarian cancers. Since BRCA2 was cloned in 1995 (Wooster et al., 1995), its involvement in the cellular response to DNA damage has become apparent (Zhang et al., 1998; Scully and Livingston, 2000; Venkitaraman, 2000). However, the biological nature of this response and the intrinsic biochemical activities of the BRCA2 protein remain enigmatic. BRCA2 is a very large protein (3418 amino acids), with a novel sequence that contains few clues to its function. One curious feature, however, is the presence of a clus§ To whom correspondence should be addressed (e-mail: s.west@

icrf.icnet.uk).

ter of eight repeat sequences (called BRC repeats; Figure 1A), encoded by exon 11, which are highly conserved between mammalian species (Bork et al., 1996; Bignell et al., 1997). It has been shown that the six more highly conserved BRC repeats (BRC1-4, BRC7, and BRC8) are involved in the interaction between BRCA2 and RAD51 (Mizuta et al., 1997; Wong et al., 1997; Chen et al., 1998b; Marmorstein et al., 1998), a protein that is essential for genetic recombination and DNA repair (Baumann et al., 1996). Additionally, in the mouse, interactions between BRCA2 and RAD51 have been reported to take place at a region located near the carboxyl terminus of BRCA2 (Mizuta et al., 1997; Sharan et al., 1997). Direct interactions between BRCA2 and RAD51 have been demonstrated by their coimmunoprecipitation from extracts prepared from normal cells, and the two proteins have been shown to colocalize at distinct nuclear foci following DNA-damaging treatments and during meiosis in germline cells (Chen et al., 1998a). Mutation of either Brca2 or Rad51 can result in embryonic lethality (Lim and Hasty, 1996; Tsuzuki et al., 1996; Connor et al., 1997; Ludwig et al., 1997; Sharan et al., 1997; Suzuki et al., 1997). The RAD51 protein possesses many biochemical activities required for homologous recombination and DNA repair, including the ability to promote joint molecule formation and DNA strand exchange between homologous DNA molecules (Benson et al., 1994; Baumann et al., 1996; Gupta et al., 1997). As a prerequisite for these activities, RAD51 binds DNA to form highly ordered nucleoprotein filaments in which the DNA is encased within a protein sheath (Benson et al., 1994; McIlwraith et al., 2000). A lack of functional BRCA2 protein leads to spontaneous gross chromosomal rearrangements and radiosensitivity (Patel et al., 1998; Yu et al., 2000), a phenotype that is indicative of defects in the recombinational repair of damaged DNA. In the work described in this paper, we have addressed the question as to whether the genetic instability associated with BRCA2 disruption may reflect, at least in part, defects in the normal activities of its partner protein, the RAD51 recombinase. Results Effect of the BRC3 Region of BRCA2 on DNA Binding by RAD51 The large size of BRCA2 has hampered attempts to obtain purified protein and analyze its biochemical properties. We therefore focused our efforts on the regions of BRCA2 (the BRC repeats) that are known to interact with RAD51, and analyzed the effect of BRC–RAD51 interactions on the in vitro properties of RAD51. To do this, we initially synthesized a 69 amino acid peptide containing the conserved 25 amino acid BRC3 motif (amino acids 1428–1452; Figure 1A). To determine whether the interaction between BRC3 and RAD51 affected the ability of RAD51 to bind DNA, we used electrophoretic mobility shift assays. In these experiments,

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Figure 1. Inhibition of RAD51–DNA Complex Formation by Peptides Corresponding to the BRC3 Region of BRCA2 (A) Schematic diagram of the BRCA2 protein, highlighting the BRC repeats that interact with RAD51. The amino acid sequence of the 69 amino acid peptide corresponding to the BRC3 repeat (amino acids 1415–1483) is indicated. The most conserved part of the BRC3 motif is shaded in gray. Three mutant peptides are also shown, and altered or deleted amino acids are indicated in red. (B) Purified RAD51 protein was incubated alone (lanes b–e) or with BRC3 (lanes g–j) prior to the addition of 32P-labeled tailed linear duplex DNA. Protein–DNA complexes were fixed and analyzed by agarose gel electrophoresis followed by autoradiography. (C) RAD51 was incubated in the absence (lane b) or presence (lanes c–i) of varying amounts of BRC3 prior to the addition of DNA.

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P-end-labeled tailed linear duplex DNA molecules were used as a substrate for RAD51 binding (McIlwraith et al., 2000). In the absence of the BRC3 peptide, RAD51 bound to the DNA, forming distinct protein–DNA complexes that were observed by agarose gel electrophoresis (Figure 1B, lanes b–e). When RAD51 was preincubated with excess BRC3 peptide before addition to the DNA, however, we found that BRC3–RAD51 interactions abolished the ability of RAD51 to bind DNA (lanes g–j). The amount of peptide required for complete inhibition was a 3- to 6-fold molar excess of peptide over RAD51 (Figure 1C).

Specificity of the BRC3–RAD51 Interaction To determine the specificity of interactions between RAD51 and BRC3, the effects of this peptide on the DNA binding activities of RAD51 homologs purified from archaea and E. coli were analyzed. As shown in Figure 2A, protein–DNA complex formation by the Rad51 protein from Archaeoglobus fulgidus (Klenk et al., 1997), which shares 45% identity with human RAD51 protein, was unaffected by the presence of a 6-fold excess of the BRC3 peptide (compare lanes c–e with g–i). Similar results were obtained in DNA binding reactions carried out using the E. coli RecA protein (data not shown). These results show that the interaction between BRC3 and human RAD51 protein is specific, and results in an inactivation of RAD51’s ability to bind DNA. Recently, a number of proteins that share sequence homology with RAD51 have been identified. These include the products of the XRCC2 (Cartwright et al., 1998; Liu et al., 1998), XRCC3 (Liu et al., 1998), RAD51B (Albala et al., 1997), RAD51C (Dosanjh et al., 1998), and RAD51D (Pittman et al., 1998) genes. Mutations in these genes cause defects in genetic recombination and DNA repair, leading to chromosome instability (Liu et al., 1998; Cui

et al., 1999; Johnson et al., 1999; Pierce et al., 1999; Thacker, 1999; Takata et al., 2000). Although these proteins share limited homology with RAD51 (usually 20%– 30% identity), we were unable to detect any evidence of interactions with BRC3, as determined using a yeast two-hybrid assay (S. Anand, A. Baillot, F.E. Benson, A. A. D., J.-Y. M., B. Randhawa, A. R. V. , S.D. Vincent, and S. C. W., unpublished observations). Similarly, BRC3 did not interact with the meiosis-specific RAD51 homolog DMC1, which shares 50% sequence identity with RAD51 (Habu et al., 1996; Yoshida et al., 1998; Masson et al., 1999), nor with RAD52 or RAD54, two other proteins also involved in recombinational repair that interact with RAD51 (Kanaar et al., 1996; Benson et al., 1998; Tan et al., 1999). As expected, in the two-hybrid assay, strong interactions were observed between BRC3 and RAD51. Thus, we conclude that the interactions of BRC3 are highly specific and restricted to RAD51 alone. Mutations within BRC3 that Mimic Cancer-Associated BRCA2 Mutations Block BRC3–RAD51 Interactions Within the eight BRC repeats of BRCA2, the most highly conserved region occurs at the sequence FxTASGK (Bignell et al., 1997). Mutations affecting this region in the BRC4 and BRC7 repeats are known to be associated with a predisposition to breast cancer. We therefore prepared a BRC3 peptide from which this seven amino acid region was deleted (Figure 1A, mutant F1428K1434). The deletion resulted in a mutant BRC3 peptide that was unable to inhibit the binding of RAD51 to DNA (Figure 2B, lanes l–n). Using two-hybrid analyses, it has been shown that a threonine to alanine mutation in the BRC1 repeat (T1011A) abrogates interactions with RAD51 (Chen et al., 1999a). When an equivalent T to A mutation was introduced into this highly conserved region of the BRC3

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Figure 2. Specificity of the BRC3–RAD51 Interaction (A) Human RAD51 (Hs RAD51) or Archaeoglobus fulgidus Rad51 (Af RAD51) was incubated with or without BRC3 peptide (24 ␮M) as indicated, and DNA binding assays were conducted as described. (B) Interactions between mutant BRC3 peptides and RAD51. RAD51 was preincubated with varying amounts of BRC3 peptide (lanes c–e), BRC3 carrying single mutations D1420Y (lanes f–h) or T1430A (lanes i–k), or BRC3 with a seven amino acid deletion ⌬F1428-K1434 (lanes l–n). The products of these interactions were analyzed for their ability to bind DNA as determined by agarose gel electrophoresis.

peptide (Figure 1A, mutant T1430A), we found that this single amino acid change was sufficient to make the peptide incapable of inhibiting DNA binding by RAD51 (Figure 2B, lanes i–k). The BRC3 repeat has been defined by sequence analysis as the region corresponding to amino acids 1415– 1483 of full-length BRCA2 protein (Bork et al., 1996; Bignell et al., 1997). We found, however, that a peptide comprising only the most highly conserved part of the BRC motif (amino acids 1428–1452; Figure 1A) was insufficient to block DNA binding by the RAD51 protein (data not shown). This observation leads us to suggest that the sequences flanking the most highly conserved region are also essential for RAD51–BRCA2 interactions. A familial mutation (D1420Y) located in the flanking sequences of BRC3 has been found in 64 cases of breast cancer (NIH Breast Cancer Information Core). A peptide containing this point mutation (Figure 1A, mutant D1420Y), however, was capable of interaction with RAD51, and excess peptide inhibited RAD51–DNA complex formation (Figure 2B, lanes f–h). The nature of this particular mutant allele and its role in tumorigenesis, therefore, remains to be determined.

BRC3–RAD51 Interactions Disrupt Nucleoprotein Filament Formation by RAD51 Interactions between RAD51 and DNA led to the formation of nucleoprotein filaments in which the DNA is stretched and underwound (Benson et al., 1994), as visualized by electron microscopy (Figure 3, upper left panel). When preincubated with the BRC3 peptide, however, RAD51 was unable to form nucleoprotein filaments, and under these conditions the DNA appeared completely naked (center left). When the BRC3 peptide was incubated with DNA in the absence of RAD51 and visualized by electron microscopy, there was no evidence of DNA binding (top right). These results show that the BRC3 peptide blocks filament formation by RAD51, and also argue against the possibility that the inhibition of RAD51’s DNA binding activity could be caused by direct interactions between the BRC3 peptide and DNA. Consistent with the band shift assays shown in Figure 2B, we found that the mutant peptides T1430A (Figure 3, center right) and F1428-K1434 (bottom right) failed to block filament formation by RAD51. When the BRC3 peptide was added to preformed RAD51–DNA complexes, we observed that the RAD51 filaments were dissociated from the DNA (data not shown). These results led us to determine whether BRC3 binds to RAD51 and blocks nucleoprotein filament formation, or whether it specifically interacts with preformed filaments to bring about their disruption. Since the competition experiment described above was carried out in standard RAD51–DNA binding buffer containing ATP, we next analyzed whether BRC3 could disrupt RAD51 filaments formed in the presence of the nonhydrolyzable ATP analogs ATP␥S or AMP-PNP. We found that stabilized RAD51 filaments, formed in the presence of the nonhydrolyzable ATP analogs, could no longer be competed off the DNA by subsequent addition of the BRC3 peptide (data not shown). Thus, disruption of the RAD51 filament by BRC3 is likely to require the turnover of the RAD51 filament in response to ATP hydrolysis, leading us to suggest that it is the initial DNA binding event that is blocked by BRC3–RAD51 interactions. Effect of BRC4 and BRC7 on Nucleoprotein Filament Formation by RAD51 We have shown that interactions between the BRC3 region of BRCA2 and RAD51 result in the conversion of RAD51 to a form that is inactive with respect to DNA binding and its function as a recombination/repair protein. To determine whether these results are representative of interactions that occur between other BRC repeats and RAD51, we synthesized peptides corresponding to the BRC4 repeat (amino acids 1511–1579) and the BRC7 repeat (amino acids 1965–2033) of BRCA2. Regions of amino acid identity between BRC3, BRC4, and BRC7 are indicated in Figure 4A. Although there is only 30% homology between BRC3 and BRC4, incubation of BRC4 with RAD51 also resulted in the conversion of RAD51 to a form that was incapable of DNA binding and nucleoprotein filament formation (Figure 4B, compare lanes b and d). In contrast, we found that equivalent molar concentrations of BRC7 only partially reduced the ability of RAD51 to form nucleoprotein filaments (lane e),

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Figure 3. Electron Microscopic Visualization of Nucleoprotein Filaments Formed by RAD51 in the Presence or Absence of BRC Peptides White arrows indicate naked duplex DNA, whereas RAD51–DNA complexes can be seen as thickened, striated nucleoprotein filaments. The black arrow indicates a ring of RAD51. All images were produced at the same magnification.

a result that was also confirmed by electron microscopy. These observations indicate that the various BRC repeats are nonequivalent in their ability to interact with RAD51 in vitro. RAD51–BRC Complex In the absence of DNA, purified RAD51 protein (37 kDa) forms multimeric ring structures that can be visualized by electron microscopy (Baumann et al., 1997; see Figure 3, black arrow). Consistent with these observations, gel filtration analysis revealed that RAD51 exhibited a broad elution profile indicative of the presence of aggregates, rings, and monomeric species (Figure 5, lane 1). To determine whether the interaction between a BRC peptide and RAD51 could affect the ability of RAD51 to self-associate, a prerequisite for nucleoprotein filament formation, we analyzed the gel filtration profile of RAD51 in the presence of the BRC4 peptide. Following interaction with BRC4, high molecular weight forms of RAD51 were not observed. Indeed, RAD51 exhibited a molecular mass consistent with the presence of a complex of a single RAD51 monomer associated with a BRC4 peptide (lane 2). When similar complexes were analyzed for the presence of the BRC4 peptide, we confirmed that the peptide was associated with the RAD51 protein in the form of a heterodimeric complex exhibiting a molecular mass of approximately 45 kDa (Figure 5, lane 4). Because these experiments were carried out using a 6-fold molar excess of peptide over RAD51, free peptide was also

observed. These data indicate that each BRC4 peptide is able to sequester one RAD51 monomer, and that when bound by BRC4, the RAD51 monomers are unable to self-associate. These results have important implications for the way that BRC peptides block nucleoprotein filament formation by RAD51. Control of RAD51 by BRCA2 In Vivo In normal cells, RAD51 and BRCA2 are known to accumulate at defined nuclear foci in response to DNA-damaging treatments (Chen et al., 1998a). The formation of these foci, however, is impaired in BRCA2-defective cell lines where reduced numbers of RAD51 foci are observed (Yuan et al., 1999; Yu et al., 2000). Consistent with the biochemical data presented in this work, these observations may indicate a cellular role for BRCA2 in the control of RAD51; for example, in a normal cell, RAD51 is held in an inactive state, but is ready to be relocalized and activated at potential sites of repair as part of the DNA damage response. To determine whether BRCA2 indeed controls RAD51 in vivo, we examined the cellular location of both BRCA2 and RAD51 in two human pancreatic epithelial cell lines: MiaPaca, which expresses full-length BRCA2, and CAPAN-1, which contains a truncated form of BRCA2 (known as BRCA2 6174delT). To do this, cells were lysed and the cytosolic and nuclear fractions separated and analyzed for the presence of BRCA2 and RAD51. In the normal cell line (MiaPaca), BRCA2 and RAD51 were found to be distributed evenly between the cytosolic

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Figure 4. Effect of BRC Peptides on RAD51–DNA Complex Formation (A) The amino acid sequences of peptides (69 amino acids long) corresponding to the BRC3 repeat (amino acids 1415–1483), the BRC4 repeat (amino acids 1511–1579), and the BRC7 repeat (amino acids 1965–2033) are indicated. Sequences that are common to all three peptides are shown in red, those shared uniquely between BRC4 and BRC3 are in green, and identities between BRC4 and BRC7 are presented in blue. (B) Purified RAD51 protein (4 ␮M) was incubated alone (lane b) or with BRC3 peptide (lane c), BRC4 peptide (lane d), or BRC7 peptide (lane e) prior to the addition of 32P-labeled tailed linear duplex DNA. All peptides were present at 24 ␮M. Protein–DNA complexes were fixed and analyzed by agarose gel electrophoresis followed by autoradiography. DNA without protein (lane a).

and nuclear fractions (Figure 6, lanes a and b). Though BRCA2 and RAD51 are primarily nuclear, the presence of RAD51 in both fractions has been observed previously (Yoshikawa et al., 2000). In contrast to the results observed with MiaPaca, extracts from the CAPAN-1 cell line revealed that the majority of RAD51 was present in the cytosolic fraction (Figure 6, lane c), and a comparatively small amount was found within the nuclear fraction (lane d). Similarly, the 200 kDa truncated form of BRCA2 (BRCA2 6174delT) present within the CAPAN-1 cells was found in the cytosolic fraction due to the loss of its nuclear localization signal (Spain et al., 1999). These data indicate that truncation of BRCA2 is associated with a defect in the normal nuclear localization of RAD51. Discussion The genomic instability phenotype associated with the BRCA2 mutation is typical of that which may be expected from cells carrying a defect in an essential DNA repair pathway (Yu et al., 2000). For example, in lower eukaryotes such as S. cerevisiae, gross chromosomal rearrangements have been observed in cells carrying mutations in genes required for DNA replication and recombination (Chen and Kolodner, 1999). Similarly, in complex eukaryotes, defects in genes required for homologous recombination result in genomic instability (Cui et al., 1999; Takata et al., 2000) and/or embryonic lethality (Lim and Hasty, 1996; Tsuzuki et al., 1996; Shu et al., 1999). Interestingly, the mutation or deletion of

nonessential recombination genes such as RAD54 (Matsuda et al., 1999), and RAD51B (Ingraham et al., 1999; Schoenmakers et al., 1999), has been shown to be associated with a variety of cancers. In vertebrates, the RAD51 recombinase is essential for the maintenance of genome integrity and for growth. Disruption of RAD51 in the mouse leads to early embryonic lethality at the egg cylinder stage, with the embryos exhibiting chromosome loss (Lim and Hasty, 1996; Tsuzuki et al., 1996). Similarly, in chicken DT40 cell lines, conditional inactivation of RAD51 was associated with loss of viability within 24 to 48 hr, and there was evidence of chromosomal aberrations and DNA fragmentation (Sonoda et al., 1998). These results serve to emphasize the importance of homologous recombination functions in normal growth, in addition to their role in the repair of DNA damage caused by external agents such as ionizing radiation. Since BRCA2 associates with RAD51, it has been suggested that the chromosomal instability observed in BRCA2-deficient cells is associated with defects in homologous recombination mediated by RAD51 (Yu et al., 2000). The work presented here both supports and extends this proposal. We have shown that peptides corresponding to the BRC3 or BRC4 repeats of BRCA2 bind RAD51 protein and inactivate it with respect to its ability to bind DNA. Inactivation is likely to be a consequence of the way that the peptides block RAD51 self-association, a property that is required for nucleoprotein filament formation. The association between the BRC repeats and RAD51 was found to be species specific, and cancer-associated mutations in the BRC3 peptide

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Figure 5. Analysis of Molecular Mass of the RAD51–BRC4 Peptide Complex RAD51 was incubated alone (lane 1) or with biotinylated BRC4 peptide (lanes 2 and 4) prior to loading onto a Superdex 200 gel filtration column. Gel filtration of the biotinylated BRC4 peptide alone is shown in lane 3. Proteins were visualized by dot blot analysis using either an anti-RAD51 antibody (lanes 1 and 2) or streptavidin-HRP (lanes 3 and 4), followed by ECL.

affected its ability to interact with RAD51. This latter result is particularly interesting in view of the fact that the eight BRC repeats are quite different; indeed, the BRC3 and BRC4 peptides (both of which interact strongly with RAD51) share only 30% identity with each other. Our results with the BRC peptides lead us to suggest that these regions provide the major interface that modulates interactions with RAD51 and exerts both positive and negative control over RAD51’s activities. Although there are eight repeats, only six have been shown to interact with RAD51, and in our own studies we find that not all interact equally; for example, the BRC7 repeat showed only a weak ability to block nucleoprotein filament assembly. The major interaction motif appears to reside in the highly conserved sequence FxTASGK, and mutations within this sequence were found to affect the RAD51–BRC interaction. This highly conserved motif, however, was not sufficient for interaction with RAD51, since a peptide comprising only the most highly conserved part of BRC3 (amino acids 1428–1452) failed to inhibit DNA binding. Given that BRCA2 contains a series of BRC repeats capable of interaction with RAD51, it is of interest to note that familial breast cancer is often associated with a single point mutation in BRCA2. Unfortunately, at the present time, we have no information relating to the three-dimensional structure of BRCA2 or the domain(s) of BRCA2 that interacts with RAD51. It is possible that the BRC repeats act independently by association with monomers of RAD51, or it is equally plausible that they act in a cooperative or concerted manner. Mutations within the BRC region that lower the affinity of one repeat for RAD51 may have wide-ranging effects throughout the RAD51 binding pocket, and may affect interactions at nearby and/or distant sites. Indeed, quite subtle changes may have gross effects in terms of the interactions of BRCA2 with RAD51, and possibly also with other components of the recombination complex that interact

with RAD51. In this respect, we note that a cancerassociated mutant allele (D1420Y) within BRC3 failed to affect the interaction with RAD51 in these in vitro assays. In related in vivo experiments (Chen et al., 1998b), it has been shown that exogenous expression of wild-type BRCA2 could complement the MMS sensitivity of CAPAN-1 cells carrying a truncated form of BRCA2. However, two familial breast cancer mutations located between BRC2 and BRC3 eliminated the ability of BRCA2 to complement CAPAN-1. These results show that the RAD51 binding repeats are necessary but not sufficient for the full function of BRCA2. What are the consequences of BRCA2–RAD51 interactions? We have shown that direct and specific interactions between the BRC3 or BRC4 repeats and RAD51 result in the conversion of RAD51 to a form that is unable to bind DNA. We suggest that this interaction effectively sequesters RAD51 in a form that is ready to be relocalized to sites of DNA damage, and thus become activated for DNA repair (Figure 7). These damaged sites, which most likely involve double-strand breaks (DSBs) formed at stalled or broken replication forks, or DSBs induced by exogenous agents, are thought to provide the signal for activation of this mammalian SOS repair response. Activation may involve the posttranslational modification of RAD51 (Yuan et al., 1998; Chen et al., 1999b) and/or occur via interactions with other repair proteins. We propose that mutations in BRCA2 that disrupt the BRCA2–RAD51 interaction may affect the maintenance of RAD51 in a form that is in this state of readiness. As a consequence, the SOS signal will go unrecognized, and repair proteins fail to assemble at the sites of DNA damage. In an extreme case, we have shown that the majority of the RAD51 within CAPAN-1 cells is found with the truncated BRCA2 (which lacks the nuclear localization signal) in the cytosolic fraction after cell lysis. Presumably, in these cells the low levels of RAD51 present within the nucleus are sufficient for proliferation, but are incapable of effecting efficient DNA repair when the cells are challenged with DNA-damaging agents.

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Figure 6. Subcellular Localization of RAD51 in BRCA2-Defective Cells Cell-free extracts were prepared from normal (MiaPaca) and BRCA2defective (CAPAN-1) cell lines and fractionated into cytosolic (Cyt) and nuclear (Nuc) fractions. These fractions were analyzed for the presence of BRCA2 and RAD51 by SDS-PAGE followed by Western blotting.

In the accompanying paper (Moynahan et al., 2001), it is shown that BRCA2-defective cell lines exhibit a significantly reduced ability to promote homologous recombination following the introduction of a defined double-strand break. An inability to promote normal repair by homologous recombination would be predicted to cause spontaneous chromosomal rearrangements including gene translocations, as observed (Patel et al., 1998; Venkitaraman, 2000; Yu et al., 2000). These genetic abnormalities, which presumably result from illegitimate homology-independent repair mechanisms (Yu et al., 2000), are likely to be a major factor in tumorigenesis. In summary, based on the data presented in this paper, we have proposed a role for BRCA2 in the control and relocalization of the RAD51 recombinase. This conclusion is supported by a number of in vivo observations. (1) Cell lines containing partial loss-of-function BRCA2 alleles exhibit a reduced frequency of chromosomal break repair by homologous recombination (Moynahan et al., 2001). (2) Mutations of BRCA2 that affect the RAD51–BRCA2 interaction are known to perturb the assembly of RAD51 nuclear foci (Yuan et al., 1999). The inability to control RAD51 and assemble a recombinosome at sites of DNA damage will give rise to the radiosensitivity associated with the BRCA2 mutation (Connor et al., 1997; Sharan et al., 1997; Morimatsu et al., 1998; Patel et al., 1998). (3) Overexpression of a GFP-BRC4 fusion protein in otherwise wild-type cells was found to block interactions between native BRCA2 and RAD51, resulting in a cell line that was significantly more sensitive to ␥ rays than control lines expressing a mutated version of the BRC4 region (Chen et al., 1999a). Indeed, cell lines containing the GFP-BRC4 fusion showed reduced numbers of radiation-induced RAD51 foci, indicating that the overexpressed BRC4 acted as a competitive inhibitor to BRCA2 by binding and inactivating RAD51. (4) The predicted consequences of BRCA2 mutation and subsequent loss of RAD51 recombinase activity, including spontaneous DNA breakage, gross chromosomal rearrangements, and mutation, have all been found in BRCA2-deficient cells. Consistent with these

Figure 7. Schematic Model for the Role of BRCA2 and RAD51 in DNA Repair (A) In a normal cell, RAD51 (green) and BRCA2 (pink) interact to form a complex with each other and with other proteins (blue). The complex may include proteins such as RAD52, RAD54, XRCC3, and RP-A. Upon DNA damage or replication fork breakdown, the complex is activated, possibly by posttranslational modification of BRCA2 or RAD51, and is recruited to the sites of DNA repair. There, RAD51 protein forms nucleoprotein filaments that, in conjunction with other repair proteins, effect double-strand break repair using the sister chromatid as a template. (B) In BRCA2 mutant cells typified by the BRCA2 truncation cell line CAPAN-1, complex formation between RAD51 and BRCA2 is disrupted, and much of the RAD51 resides in the cytoplasm along with the truncated BRCA2. The RAD51 that remains in the nucleus lacks BRCA2 control and may bind nonproductively at undamaged regions of DNA. In these cells, the introduction of double-strand breaks fails to stimulate the recruitment of BRCA2, RAD51, and other repair proteins, leading to inefficient homologous recombination and genomic instability.

observations, it has been shown that homologous recombination by gene conversion is less likely to result in chromosomal rearrangements than other RAD51independent repair processes, such as nonhomologous end-joining or single-strand annealing pathways of recombination (Richardson and Jasin, 2000). Although the model presented here appears to account for many of the phenotypic properties associated with the BRCA2 mutation, we presently know very little about the biological function of the BRCA2 protein itself, how it is regulated, or why cancer predisposition is selective for breast and ovarian tissue. Most likely, BRCA2 is modi-

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fied in response to DNA damage, possibly by phosphorylation (Venkitaraman, 1999), a regulatory mechanism that may affect both its interactions with RAD51 and the subsequent sequestration to sites of DNA damage. Defects in the signaling process in response to DNA damage may be as critical to RAD51 function as those that affect regions of the protein involved directly in BRCA2–RAD51 interactions. Further studies of the interactions between BRCA2 and RAD51 will undoubtedly shed new light on the way that BRCA2 plays its essential role in the maintenance of genomic stability and the underlying basis of genetic predisposition to breast cancer. The simplicity of the DNA binding assay described in this paper readily lends itself to the analysis of BRCA2–RAD51 interactions relevant for tumorigenesis. Moreover, the ability of selected peptides or reagents to modulate the activity of RAD51 may present new tools by which chemotherapy can be used to sensitize tumor cells to the lethal effects of ionizing radiation. Experimental Procedures Peptides Peptides were prepared by solid phase synthesis (ICRF Peptide Synthesis Unit), purified, and their sequences verified by mass spectroscopy (ICRF Protein Sequencing Laboratory). For some experiments, the BRC4 peptide contained an N-terminal biotin group with an aminohexanoic acid spacer. Proteins Human RAD51 protein (Baumann et al., 1997) and E. coli RecA protein (Eggleston et al., 1997) were purified as described. Purified Rad51 protein from Archaeoglobus fulgidus was provided by David Hall and Dale Wigley. DNA Binding Assay Linear duplex DNA (4300 bp) containing overhanging 3⬘ singlestranded tails (approximately 600 nucleotides in length) was prepared by treatment of SmaI-linearized pPB4.3 plasmid DNA (Baumann and West, 1997) with T7 gene 6 exonuclease, followed by phenol/chloroform extraction and gel purification (McIlwraith et al., 2000). The DNA was 5⬘ 32P-end-labeled using polynucleotide kinase and [␥-32P]ATP, following calf intestinal phosphatase treatment. All DNA concentrations are expressed in nucleotide residues. Equal volumes of RAD51 (in 20 mM Tris-acetate [pH 7.5], 1 mM EDTA, 0.5 mM dithiothreitol, 200 mM KOAc, and 10% glycerol) and peptide (in water) were mixed and incubated for 15 min at 37⬚C. The proteins were then supplemented with binding buffer (50 mM triethanolamine-HCl [pH 7.5], 0.5 mM Mg(OAc)2, 1 mM dithiothreitol, 2 mM ATP, and 100 ␮g/ml bovine serum albumin) and tailed duplex DNA (5 ␮M), and the incubation was continued for a further 15 min in a total volume of 10 ␮l. Protein–DNA complexes were fixed by incubation with 0.2% glutaraldehyde for 15 min at 37⬚C and were analyzed by electrophoresis through 0.8% agarose gels followed by autoradiography. For the formation of stabilized filaments, RAD51 reactions were carried out in binding buffer in which ATP was replaced by 2 mM ATP␥S or AMP-PNP. Gel Filtration RAD51 (3.2 ␮g) and biotinylated BRC4 peptide (4 ␮g) were mixed and incubated for 15 min at 37⬚C. The mixture was then supplemented with buffer (50 mM triethanolamine-HCl [pH 7.5], 0.5 mM Mg(OAc)2, 1 mM dithiothreitol, 2 mM ATP, and 100 ␮g/ml bovine serum albumin) and further incubated for 15 min at 37⬚C (20 ␮l, total volume). The proteins were then loaded onto 2.4 ml Superdex 200 PC 3.2/30 SMART columns (Pharmacia) equilibrated with the same buffer. Fractions (50 ␮l) were collected, and the elution profiles of RAD51 and BRC4 were determined by comparison with gel filtration standards (BioRad). To do this, 0.5 ␮l of each fraction was blotted onto nitrocellulose paper, and RAD51 was detected using an anti-

RAD51 polyclonal antibody (FBE1). The biotinylated peptide was observed using streptavidin coupled with horseradish peroxidase (Autogen Bioclear), followed by detection using ECL. Electron Microscopy Linear duplex molecules carrying 3⬘ single-stranded tails were produced by annealing EcoRI- and BamHI-linearized pDEA-7Z DNA (Shah et al., 1994) to pPB4.3 ssDNA (Baumann and West, 1997). Gapped, circular DNA products were purified by gel electrophoresis, electroeluted, and repurified using Qiaquick DNA purification columns (Qiagen). The DNA was then linearized with XbaI, leaving an 18-mer oligonucleotide on the complementary strand of the unique 1.3 kb 3⬘ tail. The DNA was heated at 50⬚C for 10 min to remove the oligonucleotide, and the tailed DNA was repurified. Binding reactions were carried out as described above using 4 ␮M RAD51, 24 ␮M peptide, and 5 ␮M DNA. Protein–DNA complexes were fixed using glutaraldehyde. Samples were diluted in 5 mM Mg(OAc)2 prior to uranyl acetate staining (Sogo et al., 1987). Complexes were visualized at a magnification of 20,500⫻ using a Philips CM100 electron microscope. Cellular Fractionation MiaPaca and CAPAN-1 cell lines (Goggins et al., 1996; Chen et al., 1998a; Chen et al., 1998b) were cultured in RPMI1640 media supplemented with 10% fetal calf serum. Extracts were prepared by incubation on ice for 15 min in lysis buffer containing 0.5% NP40, 50 mM Tris (pH 7.5), 25 mM NaCl, 350 mM sucrose, 3 mM MgCl2, 0.1 mM EGTA, 100 ␮M sodium vanadate, 5 mM NaF, and a cocktail of protease inhibitors (1 mM PMSF, 1.5 ␮g/ml aprotinin, 10 ␮g/ml calpain inhibitor, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin A, and 1 ␮g/ ml chymostatin). Extracts were then centrifuged at 3500 rpm for 5 min in an Eppendorf centrifuge. The cytosolic fraction was retained, while the nuclear pellet was washed by resuspension in lysis buffer minus NP40, and centrifuged again at 3500 rpm for 5 min. Cytosolic and nuclear fractions were diluted to an equal volume with SDS sample buffer and analyzed for the presence of BRCA2 and RAD51 using SDS-PAGE and Western blotting. The top half of the blot was analyzed with a polyclonal antibody raised against the BRC3 peptide (SWE28), and the bottom half with a rabbit polyclonal antibody raised against RAD51 (FBE1). Acknowledgments We thank Dale Wigley for generously providing us with A. fulgidus Rad51, Dhira Gadhia, Nicola O’Reilly, and Hans Hansen for peptide synthesis and analysis, Ruth Peat and colleagues for cell culture, and Maria Jasin, Susan Critchlow, and other members of our laboratory for providing insightful comments. This work was supported by the Imperial Cancer Research Fund (S. C. W.), the Human Frontiers Science Program (S. C. W. and A. S.), the Swiss National Foundation (A. S.), the Swiss-British Council Joint Research Program (S. C. W. and A. S.), the Cancer Research Campaign (A. R. V.), and the Medical Research Council (A. R. V.). A. R. V. holds a professorship generously endowed by the late Dr. F. A. Zoellner. J.-Y. M. was supported by a fellowship from the National Cancer Institute of Canada. Received August 21, 2000; revised January 3, 2001. References Albala, J.S., Thelan, M.P., Prange, C., Fan, W., Christensen, M., Thompson, L.H., and Lennon, G.G. (1997). Identification of a novel human RAD51 homolog, RAD51B. Genomics 46, 476–479. Baumann, P., and West, S.C. (1997). The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A. EMBO J. 16, 5198–5206. Baumann, P., Benson, F.E., and West, S.C. (1996). Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766. Baumann, P., Benson, F.E., Hajibagheri, N., and West, S.C. (1997). Purification of human Rad51 protein by selective spermidine precipitation. Mutat. Res. 384, 65–72.

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