Homologous Repair of DNA Damage and Tumorigenesis: the BRCA Connection

Homologous repair of DNA damage and tumorigenesis: the BRCA connection

Maria Jasin*,1

1Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

Homologous recombination has been recognized in recent Tirkkonen et al., 1997; Turner et al., 2002; Xu et al., years to be an important DNA repair pathway in 1999), are sensitive to DNA damaging agents (Bhatta- mammalian cells, for such damage as chromosomal charyya et al., 2000; Connor et al., 1997; Gowen et al., double-strand breaks. Cells mutated for the genes 1998; Kraakman-van der Zwet et al., 2002; Morimatsu involved in the hereditary breast and ovarian cancer et al., 1998; Moynahan et al., 2001a; Scully et al., 1999; susceptibility syndromes, i.e. BRCA1 and BRCA2, show Sharan et al., 1997; Shen et al., 1998; Zhong et al., defects in DNA repair by homologous recombination, 1999), including ionizing radiation and cross-linking implicating this repair pathway in protecting individuals agents, and have elevated mutation rates (Kraakman- against tumorigenesis. This review summarizes recent van der Zwet et al., 2002; Tutt et al., 2002). During S advances in our understanding of BRCA1 and BRCA2 in phase or upon treatment of cells with DNA damaging DNA repair, as well as insight into these proteins agents or replication inhibitors, both proteins are in gleaned from structure determination of domains of these nuclear foci along with several other proteins involved proteins and the broader evolutionary conservation than in the damage response, including the DNA repair previously appreciated. protein RAD51 (Chen et al., 1998a; Scully et al., Oncogene (2002) 21, 8981 – 8993. doi:10.1038/sj.onc. 1997a,b). Nuclear focus formation, at least for 1206176 BRCA1, is affected by proteins involved in the damage response (Bassing et al., 2002; Celeste et al., 2002; Keywords: BRCA1; BRCA2; DNA repair; homologous Wang et al., 2002). In addition, BRCA1 is phosphory- recombination; RAD51 lated in a cell cycle manner (Ruffner and Verma, 1997; Scully et al., 1997a) and after genotoxic stress in a manner dependent upon the DNA damage signaling Introduction proteins ATM, ATR, and CHK2 (Cortez et al., 1999; Gatei et al., 2000, 2001; Lee et al., 2000; Tibbetts et al., Individuals that carry a germline mutation in BRCA1 2000), and Brca1 or Brca2 mutation in mice leads to or BRCA2 are predisposed to breast, ovarian, and a the induction of the p53-p21 DNA damage checkpoints few other cancers (Scully and Livingston, 2000; Welcsh (Connor et al., 1997; Hakem et al., 1996; Patel et al., and King, 2001). Following the paradigm of tumor 1998; Suzuki et al., 1997; Xu et al., 2001b). suppressor genes, one mutated allele of BRCA1 or A major challenge is to determine the molecular BRCA2 is inherited and then somatic mutation occurs roles of BRCA1 and BRCA2 in the cellular response to to alter the second allele, such that tumors invariably genotoxic stress. The association of these proteins in contain two mutant alleles. The lifetime risk associated nuclear foci with RAD51, the homolog of the with inheritance of a mutated BRCA1 or BRCA2 prokaryotic RecA protein, implicates these proteins in allele, while significant, has nevertheless proved difficult homologous recombination, a DNA repair pathway in to estimate precisely (Begg, 2002). which a damaged DNA molecule uses a homologous BRCA1 and to a lesser extent BRCA2 have been sequence to direct repair (Jasin, 2001). RAD51 directly implicated in diverse cellular processes (Scully and interacts with peptides derived from BRCA2 (Mizuta Livingston, 2000; Welcsh and King, 2001), including et al., 1997; Sharan et al., 1997; Wong et al., 1997). recently the maintenance of X chromosome inactiva- Thus far, however, a direct protein-protein interaction tion (Ganesan et al., 2002). However, several lines of between BRCA1 and RAD51 has not been reported, investigation strongly implicate both of these nuclear suggesting that the association is indirect. proteins in the DNA damage response. BRCA1 and Because multiple genetic hits are necessary for BRCA2 mutant cells exhibit a high degree of tumorigenesis, individuals that carry germline muta- spontaneous and induced chromosome aberrations tions in DNA damage response genes are particularly (Kraakman-van der Zwet et al., 2002; Moynahan et cancer prone, due to the hypermutability of their cells al., 2001a; Patel et al., 1998; Shen et al., 1998; (Vogelstein and Kinzler, 1998). Thus, like other DNA repair pathways homologous recombination may guard cells from becoming tumorigenic. As yet, there is scant *Correspondence: M Jasin; E-mail: [email protected] evidence to support this based on the analysis of widely BRCA1, BRCA2, and homologous DNA repair M Jasin 8982 conserved recombination genes, i.e., RAD51 itself, the originally identified through a two-hybrid screen with RAD51 paralogs, RAD52, and RAD54 (Pierce et al., BRCA1 (Wu et al., 1996). Although a large number of 2001). However, the interaction of BRCA proteins with proteins have been found to interact with BRCA1, RAD51, especially the direct interaction with BRCA2, what is notable is that most or all of the BRCA1 in the strongly suggests a connection between this DNA cell appears to be in a complex with BARD1 (Yu and repair pathway and tumor suppression. This review Baer, 2000). Like BRCA1, BARD1 contains an N- summarizes recent developments in our understanding terminal RING domain and two C-terminal BRCT of the structure and biochemical activities of the domains. However, the central region of BARD1, BRCA proteins and then reviews evidence which which contains a set of ankyrin repeats not found in implicates BRCA1 and BRCA2 in DNA repair by BRCA1, is significantly smaller than that of BRCA1, homologous recombination. Mouse tumor models and such that the protein has a total length of 777 amino characteristics of the human disease are discussed in acids. Perhaps because a larger fraction of BARD1 is the subsequent review (Moynahan, 2002). composed of these conserved domains, its overall sequence conservation between human and mouse is greater (70%) than that of BRCA1. BRCA1 and BRCA2 basics: sequence conservation, The RING motif of BRCA1 was immediately structure determinations, and biochemical activities recognizable when the gene was cloned (Miki et al., 1994). RING motifs are approximately 50 amino acids Notable about the BRCA1 and BRCA2 proteins are and consist of 8 Cys and His residues (Cys3HisCys4) their poor sequence conservation across mammalian which bind two Zn2+ atoms in an interleaved fashion. species. For example, the sequence identity of human The RING motifs of BRCA1 and BARD1 are each BRCA1 with mouse Brca1 is 56%, and human BRCA embedded within the larger RING domain of about 90 with mouse Brca2, 57%. This contrasts with other amino acids which is necessary for BRCA1-BARD1 genomic caretaker tumor suppressor genes, such as heterodimer formation (Meza et al., 1999). The MSH2 or XPA, where the identity between human and solution structure determination clarified the role of mouse is much higher (92% and 86%, respectively), or these extra 40 amino acids (Brzovic et al., 2001b). In even with p53, where the identity is 77%. Strikingly, each protein they form antiparallel a-helices, which the RAD51 strand exchange protein, which is in interact in the heterodimer to form a four-helix bundle nuclear foci with BRCA1 and BRCA2, is 99% (Figure 1). The RING motifs from BRCA1 and identical between human and mouse. Despite the lack BARD1 extend from the same side of the four-helix of strong sequence conservation, the human BRCA1 bundle, and although they do not pack against each gene can rescue the embryonic lethality of Brca1 other, they are in close proximity. mutant mice (Chandler et al., 2001; Lane et al., 2000), implying a functional conservation of the protein. Similarly, the human BRCA2 gene can apparently rescue DNA damage sensitivities of a hamster Brca2 mutant cell line (Kraakman-van der Zwet et al., 2002). Even with the overall poor sequence conservation, BRCA1 and BRCA2 are characterized by regions of higher sequence conservation. BRCA1 has two such conserved regions, a RING domain at the N-terminus and a BRCT repeat domain in the C-terminus. These two regions account for only 300 amino acids of the 1863 amino acid BRCA1 protein, however, a mere 16% of the protein. The 3418 amino acid BRCA2 has a conserved C-terminal region, but also conserved BRC motifs located in the central portion of the protein. Structural determinations have been made for several of these domains in the past year. These structures will be discussed, as well as biochemical activities that have been ascertained for these domains or other regions of the proteins.

BRCA1 Figure 1 Schematic representation of the BRCA1-BARD1 het- Structure of the BRCA1-BARD1 heterodimeric RING erodimeric RING domain, after Brzovic et al., 2001b. The pep- domain: BRCA1 amino acids 8 – 96 tides of the RING domain interact at a four-helix bundle, formed by two anti-parallel a-helices from each of BRCA1 and The BRCA1 RING domain forms a heterodimeric BARD1. The RING motifs themselves, which extend from the complex with the RING domain of BARD1, a protein helices, each bind two Zn2+ atoms

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8983 Cancer-predisposing missense mutations are found at ubiquitination, raising the possibility that the BRCA1- five of the Cys residues in the BRCA1 RING motif BARD1 heterodimer may also undergo auto-ubiquiti- (BIC Breast Cancer Information Core webside http: // nation. This would seem to be counter to the www.nchgr.nih.gov/dir/Intramural_research/Lab_trans- observation that the two proteins stabilize each other fer/Bic/index.htm 1). Based on biochemical studies of (Chen et al., 2002; Hashizume et al., 2001; Joukov et the RING domain peptides, these mutations have al., 2001). Nevertheless, using an N-terminal fragment differential effects; some completely disrupt folding of of BRCA1 (amino acids 1 to 639), the BRCA1- the peptide, while others, including the common C61G BARD1 heterodimer has been found to undergo mutation, have a more subtle local structural effect auto-ubiquitination in vitro, such that both the BRCA1 (Brzovic et al., 2001a). A number of unclassified fragment and BARD1 were ubiquitinated (Chen et al., variants distributed throughout the RING domain, 2002). A shorter N-terminal BRCA1 (amino acids 1 – including the dimerization helices, have been reported 300)-BARD1 was observed to be ubiquitinated when to the BIC database but their biochemical conse- tested in vivo as well. Lysine 48-linked polyubiquitin quences are not known. As yet no clear evidence chains are signals for degradation of the protein linked implicates BARD1 disruption in tumorigenesis, to ubiquitin; thus, the requirement for this amino acid although a few tumor-derived mutations have been in ubiquitin was tested in vitro. The N-terminal identified (Thai et al., 1998). BRCA1-BARD1 heterodimer efficiently utilized ubiquitin K48R, suggesting that the ubiquitin chain signals something other than, or possibly in addition to, Functional studies of the BRCA1-BARD1 RING domain: degradation (Chen et al., 2002). Ubiquitin protein ligase activity Although the Ub ligase activity may be important RING proteins were recognized several years ago to for auto-ubiquitination, the BRCA1-BARD1 hetero- have ubiquitin (Ub) protein ligase activity (i.e., E3 dimer could have other substrates as well. BRCA1 is in activity), facilitating the transfer of ubiquitin from a nuclear foci with a number of other proteins, including Ub conjugating enzyme to substrate proteins. An N- a phosphorylated form of the histone variant H2AX terminal fragment of BRCA1 (amino acids 1 – 788) was (Paull et al., 2000). Since histone H2A and variant tested and found to possess such an activity, and this H2AX are known to be mono-ubiquitinated, the activity was Zn2+dependent (Lorick et al., 1999). A proteins were tested in the N-terminal BRCA1-BARD1 truncated RING domain of BRCA1, encoding up to in vitro ubiquitination reaction and found to be amino acid 78, was also found to possess Ub ligase ubiquitinated (Chen et al., 2002). Other substrates activity, and this activity was compromised by have also been proposed, including the FANCD2 mutation of conserved Cys residues, including C61G protein, which undergoes mono-ubiquitination in a (Ruffner et al., 2001). BRCA1-dependent manner in vivo in response to Given that BRCA1 forms heterodimers with ionizing radiation (Garcia-Higuera et al., 2001). BARD1, Ub ligase activity for the heterodimer has Heterodimerization of BRCA1 with BARD1 may also recently been examined, using N-terminal frag- have an additional consequence in addition to ments of BRCA1 (amino acids 1 – 342 or 1 – 772) promoting ubiquitin ligase activity. Nuclear localiza- (Hashizume et al., 2001). Heterodimeric N-terminal tion signals have been mapped to the central region of BRCA1-BARD1 was found to have significantly higher BRCA1; however, a nuclear export signal has been activity than BRCA1 or BARD1 by themselves. The mapped to the RING domain of BRCA1, which tumor-derived C61G mutation again completely abol- opposes the action of the nuclear localization signal ished the activity of the heterodimer. Interestingly, (Rodriguez and Henderson, 2000). Binding to BARD1 mutation of the cognate Cys in BARD1 (C83G) had masks the export signal, so that BRCA1 isoforms that little effect on the activity of the heterodimer do not contain the nuclear localization signal can still (Hashizume et al., 2001). These experiments were localize to the nucleus (Fabbro et al., 2002). performed using the Ub conjugating enzyme UbcH5, In addition to BARD1, other proteins have been which by analogy with the cCbl-UbcH7 complex, has reported to bind the BRCA1 RING domain (Welcsh et been proposed to bind to the RING motif of BRCA1 al., 2000), including BAP1, which interestingly is a Ub at a region that differs from the RING motif of hydrolase (Jensen et al., 1998). BARD1 (Brzovic et al., 2001b). Thus, the BARD1 C83G mutation may not affect the interaction of the Structure of the BRCA1 BRCT domain: BRCA1 amino BRCA1-BARD1 heterodimer with UbcH5. acids 1646 – 1859 Major questions remain regarding the functional consequence of the Ub ligase activity of the BRCA1- Sequence analysis of BRCA1 revealed that its C- BARD1 heterodimer and the biologically relevant terminus contained a repeated motif of approximately substrates. The initial report of Ub ligase activity for 100 amino acids, which were termed BRCT repeats the BRCA1-BARD1 heterodimers detected free ubiqui- (BRCA1 C-terminal repeats) (Callebaut and Mornon, tin polymers or ubiquitin polymers attached to another 1997; Koonin et al., 1996). The recognition of this molecule, possibly a protein which could be targeted domain in BRCA1 quickly led to the identification of for degradation by the 26S proteasome (Hashizume et its widespread presence in a number of nuclear proteins al., 2001). Other RING domain proteins undergo auto- involved in DNA repair. The BRCT domain and

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8984 adjacent sequences have also been implicated in al., 2001). Xenopus BARD1 was described in the same transcriptional activation and chromatin unfolding report (Joukov et al., 2001). (Monteiro, 2000; Ye et al., 2001). The greater divergence of the chicken sequence The crystal structure of the human and rat BRCA1 allowed an identification of more conserved patches BRCT domains have recently been solved, revealing of amino acids in the middle part of the protein similarities of the two BRCA1 BRCT repeats to each (Orelli et al., 2001), many of which also appear to other as well as to BRCT repeats found in other be conserved in the Xenopus protein. These types of proteins (Joo et al., 2002; Williams et al., 2001). Each phylogenetic comparisons may shed light on some of repeat has a central core of four parallel b strands the unclassified variants found in BRCA1, by hinting flanked by a helices. The two repeats are arranged in a at which variants are most likely to alter protein head to tail fashion with a rotation and they pack function. Functional studies have not as yet been against each other at the helices and at a linker region reported for the chicken protein; however, antisense at the interface of the two repeats. experiments were performed in two-cell Xenopus Cancer predisposing truncating mutations are found embryos to determine the effect of loss of BRCA1 throughout this region (BIC). A large number of or BARD1 in frogs (Joukov et al., 2001). Depletion missense mutations are also found, although most of was observed at the onset of gastrulation, with these are unclassified variants. The few known cancer developmental abnormalities observed several hours predisposing missense mutations in this region have later at the tadpole stage. Gross phenotypic been predicted to interfere with packing of the BRCT abnormalities were detected, with malformations in repeats to each other (Williams et al., 2001). Thus, several tissue types likely as a result of proliferation when tested by assaying the sensitivity of the peptides defects. to limited proteolysis, A1708E and, to a lesser extent, More recently, Caenorhabditis elegans BRCA1 and M1775R were found to destabilize the folding of the BARD1 orthologs have been identified, BRC-1 and BRCT repeats (Williams et al., 2001). BRD-1, respectively (SJ Boulton, personal communication). Phenotypic analysis of these two genes suggest a shared role in DNA repair, as with the human DNA binding of BRCA1 proteins. Interestingly, a BRCA1-related protein in The BRCA1 RING motif was originally considered to Arabidopsis has also been identified, which has 24% be a potential DNA binding domain because of the identity in the RING domain to the human protein similarity to Zn fingers, which are known DNA and 29% identity in the BRCT, although this protein binding motifs (Lovering et al., 1993). However, direct has an N-terminal extension with other conserved tests for DNA binding indicated this domain does not protein motifs (Joukov et al., 2001). It was noted that bind DNA (Meza et al., 1999), consistent with this the degree of identity between the Arabidopsis protein domain being involved instead in protein – protein with human BARD1 was similar to the identity with interactions as described above. Conflicting data exists human BRCA1, perhaps reflecting a common evolu- on the BRCT domain, as one report finds evidence of tionary ancestor for BRCA1 and BARD1 which is still DNA binding by this domain (Yamane et al., 2000), found in this organism. No clear fungal BRCA1 whereas a second report does not (Paull et al., 2001). homologs have been reported; however, BRCT- More recently full-length BRCA1 protein and an containing proteins that share features with BRCA1 internal peptide (amino acids 452 – 1079) have been have been noted, for example Crb2 in the fission yeast found to bind DNA (Paull et al., 2001). Binding was (Caspari et al., 2002) nonspecific with regards to sequence, but preferential to branched DNA structures and long DNA molecules. BRCA2 Electron microscopy with the BRCA1 peptide indicates DNA loop formation, with multiple peptide molecules BRC repeats and the conserved C-terminal domain bound at the base of the loop. The significance of this finding is as yet unclear. Sequence comparison with proteins in the database did not reveal significant similarities of BRCA2 to other proteins when it was originally cloned (Tavtigian et al., Non-mammalian BRCA1 homologs 1996). Neverthelesss, dot matrix analysis comparing Until recently, BRCA1 homologs had only been BRCA2 with itself uncovered an internally repeated identified in mammalian species, including rat and sequence, termed a BRC motif, found eight times dog, in addition to mouse and human. Last year within the middle third of the BRCA2 protein (Bork et chicken and frog proteins were identified as well. al., 1996). A BRC repeat consensus core sequence Chicken BRCA1 shows 33% identity overall with the spanning 26 amino acids has been identified by human protein, although 67% identity in the RING comparing multiple mammalian species, although each domain (compared with 590% identity between repeat may span a larger segment of approximately 70 mammalian species) and approximately 60% identity amino acids (Bignell et al., 1997). However, only five of in the BRCT domains (Orelli et al., 2001). The Xenopus the repeats (BRC1, 3, 4, 7, 8) conform well to the laevis BRCA1 protein has a similar degree of identity consensus. The spacing of the repeats is variable, with the human protein in these domains (Joukov et ranging from 80 to more than 300 amino acids when

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8985 considering a fixed amino acid position within the two in Arabidopsis and one in rice (Warren et al., repeats, such that the entire BRC repeat region spans 2002), as well as more recently C. elegans (SJ Boulton, more than 1000 amino acids. Across vertebrate species, personal communication). While the C-terminal region including chicken (Takata et al., 2002; Warren et al., is conserved in these BRCA2-related proteins, they 2002), individual repeats are more similar to each other have a variable number of BRC repeats. For example than to other repeats, and the spacing is reasonably only four BRC repeats have been identified in the well-maintained. In addition to the BRC repeats, two Arabidopsis protein. However, at the extremes, 15 BRC other short regions of particular conservation have repeats have been found in the trypansome protein, been noted in this region of the protein, one between although none at all in the E. cuniculi protein. Notably BRC1 and BRC2 and the other just downstream of absent from these BRCA2-related proteins are signifi- BRC8 (Bignell et al., 1997; Sharan and Bradley, 1997; cant N-terminal sequences upstream of the BRC Takata et al., 2002; Warren et al., 2002), although their repeats, so in general they are much smaller than the significance is not as yet clear. vertebrate proteins. Although a Rad51 interacting domain was first Despite the lack of identifiable homologs in both identified at the C-terminus of the mouse Brca2 protein budding and fission yeast, a BRCA2 homolog was (Mizuta et al., 1997; Sharan et al., 1997), the BRC recently identified in the yeast-like fungus Ustilago repeat region is considered to be the major RAD51 maydis in a screen for DNA repair mutants (Kojic et binding region of human BRCA2 (Chen et al., 1998b; al., 2002). The homolog, termed BRH2, encodes a 1075 Wong et al., 1997). Six of the eight BRC repeats amino acid protein with only a single identified BRC interact with RAD51 in yeast two-hybrid assays, repeat. The C-terminus shares more extensive similarity although the remaining two repeats can interact with with the mammalian proteins, with an approximately RAD51 in in vitro assays (Wong et al., 1997). A 200 amino acid region having 38% identity with structure determination has recently been made for an human BRCA2 in the region that interacts with DSS1. isolated BRC peptide bound to the RecA-homology domain of RAD51 (Pellegrini et al., 2002). The BRC Structure of the BRCA2 C-terminal domain with peptide mimics a motif in RAD51 at the monomer- DSS1: DNA binding monomer interface of RAD51 and thus can presumably control the assembly of the RAD51 nucleoprotein The structure of the BRCA2 C-terminal domain has filament (see below). recently been solved bound to DSS1 (Yang et al., Following the BRC repeat region is the much more 2002). Co-expression of the two proteins was found to highly conserved C-terminal region, which in human be necessary to obtain soluble BRCA2 protein. Limited and mouse share 77% identity (Mcallister et al., 1997; proteolysis was used to optimize the domain bound- Sharan and Bradley, 1997). Chicken BRCA2 also aries for crystallization, with a 736 amino acid BRCA2 shares much more conservation with the mammalian peptide from mouse found to give the best diffracting proteins in the C-terminal third of the protein than it crystals (approximately residues 2400 – 3110). Four does with the middle third of the protein containing globular domains are present in the BRCA2 structure the BRC repeats or the approximately 1000 amino acid in a linear array, along with an unusual fifth domain N-terminus (Takata et al., 2002; Warren et al., 2002). extending as a tower-like structure from one of the The C-terminal region binds the 70 amino acid DSS1 globular domains (Figure 2). Three of the four protein, as identified by two-hybrid analysis (Marston globular domains, each of approximately 110 amino et al., 1999), and its structure has recently been solved acids, form OB folds (OB, oligonucleotide/oligosac- in complex with DSS1 ((Yang et al., 2002); see below). charide-binding), which are found in single-stranded The vertebrate BRCA2 homologs, especially the DNA binding proteins like the mammalian RPA more distant chicken BRCA2, provide sequences for protein. The tower domain, which extends from the comparative analysis to attempt to assign unclassified middle of the three adjacent OB folds, consists of two variants of human BRCA2 (Warren et al., 2002). long a-helices capped by a three-helix bundle, the latter Known pathogenic and polymorphic variants show a of which has a structure similar to DNA binding clear difference in distribution between conserved and domains found in other proteins such as Hin non-conserved amino acids, suggesting that this recombinase. The other globular domain, located N- approach may have a good predictive value to classify terminal to the OB folds, primarily consists of a- variants, support of which can be gained by comple- helices. The small protein DSS1 binds in an extended mentary functional approaches. conformation to the helical domain and the first two OB folds, in part tunneling through the BRCA2 structure. DSS1 has clusters of both hydrophobic and Non-vertebrate BRCA2 homologs acidic residues and binds to a portion of BRCA2 that Although BRCA2 homologs have not been identified is rich in basic, aromatic, and hydrophobic residues, in budding or fission yeast, BRCA2-related proteins leading to the speculation that in the absence of DSS1 have been identified in diverse eukaryotic species, this region of BRCA2 may have the potential to including puffer fish, mosquito, several intracellular interact with single-stranded DNA (Yang et al., 2002). parasites, including trypanosome and the microspir- Given the structural similarity to RPA, the BRCA2 idium Encephalitozoon cuniculi, and plants, including C-terminal domain, in complex with DSS1, was

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8986

Figure 2 Schematic representation of the BRCA2 C-terminal domain in complex with DSS1 and single-stranded DNA, after Yang et al., 2002. Two of the three OB folds, which are similar to domains found in RPA, bind single-stranded DNA in the structure determination. At the top of the Tower, the three-helix bundle (3hb), comprised of three short a-helices, may form a second DNA binding domain, possibly for double-stranded DNA. Crystallization was facilitated by co-expression of the BRCA2 C-terminal domain with the small peptide DSS1. The N-terminus of the BRCA2 peptide is at the start of the helical domain on the far right; the C-terminus is at the end of the OB3 domain on the far left

assayed for DNA binding (Yang et al., 2002). Similar most extensive contact with DNA have been found to to other single-stranded DNA binding proteins, single- be mutated in cancers. Overall, a large number of stranded DNA, either mixed sequence or oligo(dT) or tumor derived missense mutations in the BIC database oligo(dC), was found to bind to the BRCA2 peptide, map to the BRCA2 C-terminal domain, including four but double-stranded DNA or oligo(dA) or oligo(dG), in the tower domain, which are frequently reported to which tend to form secondary structures, did not bind. be mutated. Thus, the structure of the BRCA2 C- With oligonucleotides up to approximately 20 nucleo- terminal domain, together with biochemical assays tides, a single gel shift was seen; however, with longer such as DNA binding, provides a context in which to oligonucleotides (up to 60 nucleotides), multiply-shifted consider the eﬀect of tumor-derived mutations. species were observed. When the tower domain was deleted, only a single species was detected even with long oligonucleotides, giving rise to the speculation Molecular analysis of homologous recombination in that long oligonucleotides, which may form secondary BRCA1 and BRCA2 mutant cell lines structures, are able to bind to the tower, perhaps at the three-helix bundle. The association of BRCA1 and BRCA2 with RAD51 The structure of the BRCA2 C-terminal domain in implicated these proteins in RAD51-mediated repair of complex with DSS1 was also solved bound to a short DNA damage by homologous recombination. Homo- oligonucleotide (Yang et al., 2002). The best crystals logous recombination, in which an identical or similar were obtained with the tower domain deleted. The DNA sequence directs the repair of damaged DNA in single-stranded DNA binds in the second two OB folds a gene conversion mechanism, can be a very precise (Figure 2), in a manner similar to RPA. Of interest, form of DNA repair because it can restore the original three of the residues in the OB fold that makes the sequence present before the damage occurred (Jasin,

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8987 2001). Damage that is repaired by homologous protein, such that the RING and BRCT domain recombination can arise from exogenous sources or coding sequences are intact. A related mutation has from endogenous processes. By analogy with E. coli, also been constructed in which all of exon 11 was homologous recombination in mammalian cells may deleted by lox-Cre recombination (Xu et al., 1999). play an important part in every cell cycle by repairing This Brca1D11 mutation also leads to expression of the damage arising during DNA replication (Cox et al., exon 11-deleted splice variant, which encodes a 92 kD 2000). DNA lesions known to be recombinogenic in peptide (Huber et al., 2001). The participation of this mammalian cells include double-strand breaks, which peptide in the DNA damage response has been will stimulate homologous recombination by several investigated. Unlike the intact 220 kD Brca1 protein, orders of magnitude (Rouet et al., 1994). Homologous a shift in protein mobility in response to DNA damage repair of double-strand breaks occurs preferentially is not detected for the 92 kD Brca1 peptide, suggesting between identical sister-chromatids, with chromosome that it is not phosphorylated (Huber et al., 2001). homologs or sequence repeats on heterologous chro- Nevertheless, like the intact protein, the 92 kD peptide mosomes apparently used much less frequently localizes to nuclear foci upon DNA damage (Huber et (Johnson and Jasin, 2000, 2001). Double-strand breaks al., 2001). Despite this, Rad51 localization to nuclear can also be repaired by nonhomologous end-joining foci upon DNA damage is impaired ((Huber et al., (NHEJ), a process in which DNA ends are rejoined 2001); see also Bhattacharyya et al. (2000)), and the 92 with little or no sequence homology (van Gent et al., kD peptide does not associate with Rad51 as assayed 2001). by co-immunoprecipitation (Huber et al., 2001). The Double-strand break repair has recently been lack of association of the 92 kD peptide with Rad51 is examined at the molecular level in a number of consistent with previous mapping studies which BRCA1 and BRCA2 mutant cell lines, including concluded that Rad51 interaction was localized to a human tumor lines and mouse embryonic stem cell region of exon 11 (Scully et al., 1997b), and it is also lines with targeted mutations. (See Moynahan, 2002, consistent with the observed recombination defect. for a review of phenotypes of mice with targeted Proteins involved in homologous recombination in mutations). Both BRCA1 and BRCA2 mutant lines actively dividing cells are often also involved in meiotic have been reported to have impaired homologous recombination. Consistent with a role in meiosis, recombination. Some recent studies also implicate BRCA1 colocalizes with RAD51 during meiosis on BRCA1 in NHEJ, although others have not found a chromosome axes prior to homolog synapsis, and in role for BRCA1 in this process, as will be discussed in males remains associated with the unsynapsed X a separate section. In addition, evidence has been chromosome (Scully et al., 1997b). A role specifically provided that BRCA2 suppresses a third double-strand in meiotic recombination is supported by the prophase I break repair pathway, termed single-strand annealing, meiotic arrest observed in Brca1D223-763/D223-763 p537/7 which can occur between sequence repeats. males (Cressman et al., 1999). Interestingly, ovaries of the one female reported in this study had growing follicles, suggesting that Brca1 may not be as stringently BRCA1 required for early meiotic events in female germ cells as in males (Cressman et al., 1999). Male but not female The Brca1 mutant mouse embryonic stem cell line infertility has been observed in a second line of Brca1 Brca1D223-763/D223-763 has been found to have a fivefold hypomorphic mice, in this case carrying a Brca1 reduction in homologous repair of a chromosomal truncation mutation (Ludwig et al., 2001). The gender double-strand break compared to wild type cell lines differences may indicate a differential role in meiotic (Moynahan et al., 1999) and a severely reduced level of recombination; alternatively, male-specific events invol- gene targeting (Moynahan et al., 1999, 2001a; ving the X-Y chromosome pair may have a particular Snouwaert et al., 1999). The Brca1D223-763/D223-763 requirement for Brca1 (see also Ganesan et al., 2002). mutant is clearly a hypomorph (partial loss of function), as gauged by the longer embryonic survival of mice containing this mutation compared with null BRCA2 alleles (Gowen et al., 1996) and the recovery of some viable mice on a p53 background (Cressman et al., Mouse cell lines with targeted mutations in the Brca2 1999). Thus, it is possible that the fivefold reduction gene also have been reported to have homologous reflects a partial loss of function, and that complete recombination defects. The Brca2lex1/lex2 embryonic loss of Brca1 leads to a more profound effect on stem cell line has two different alleles of Brca2: the homologous repair, although this has yet to be tested. lex1 allele in which the terminal exon 27 is deleted by The Brca1D223-763 mutation deletes the 5’ portion of gene targeting, resulting in a truncation at amino acid exon 11 (Gowen et al., 1996). The hypomorphic nature 3139 of the 3328 amino acid mouse Brca2 protein and of this mutation is thought to be due to the expression the lex2 allele in which exon 27 is deleted together with of a splice variant in which exon 11 is entirely skipped a portion of exon 26 at amino acid 3088 (Morimatsu et (Snouwaert et al., 1999), such that exons 10 and 12 are al., 1998). The set of BRC repeats in the central spliced together at amino acids 222 and 1328. This portion of the protein is therefore left intact in both splicing event maintains the reading frame of the alleles. The C-terminus of the protein was targeted for

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8988 disruption in this study, as it was the first portion of for homologous recombination, the Brca2Tr/DEx27 cell the protein recognized to interact with Rad51 (Mizuta line was found to have a 42% reduction in homologous et al., 1997; Sharan et al., 1997). repair of a double-strand break compared with the When tested for repair of a chromosomal double- control Brca2Tr/Ex27+ cell line in which the Brca2Ex27+ strand break, a fivefold reduction in homologous repair allele of Brca2 is wild-type except for an epitope tag. was observed in the Brca2lex1/lex2 cells (Moynahan et In addition to homologous repair by gene conversion al., 2001b). As with the Brca1D223-763/D223-763 mutant, and non-homologous repair by NHEJ, another the Brca2lex1/lex2 mutant is certainly a hypomorph, outcome of double-strand break repair is a deletion since, unlike mice with Brca2 null alleles, viable mice involving a sequence repeat. Such deletions can arise with a Brca2 exon 27 deletion can be obtained through annealing of single stranded DNA generated (McAllister et al., 2002). Thus, the moderate fivefold at the repeat rather that a Rad51-mediated gene reduction may reflect a partial loss of function, such conversion process (Paˆques and Haber, 1999). Deletion that a Brca2 null cell line would have a more profound events were assayed in the Brca2Tr/DEx27 mutant and defect in homologous repair. Interestingly, although found to be about threefold elevated compared with this fivefold reduction in homologous repair was the the Brca2Tr/Ex27+ cell line (Tutt et al., 2001). A recent same as that observed in the Brca1D223-763/D223-763 study examining spontaneously arising deletion events mutant, the fold-reduction in gene targeting Brcalex1/ also found an increase in deletion events in a BRCA2 lex2 mutant is much milder as compared with the mutant (Larminat et al., 2002). This contrasts with Brca1D223-763/D223-763 mutant (i.e., twofold vs 16-fold results from the Brca1D223-763/D223-763 mutant, in which using the same targeting vector; (Moynahan et al., deletion events following a double-strand break were 2001a,b)). Thus, although both proteins have a role in decreased rather than increased (Moynahan et al., promoting homologous recombination, their role does 1999). Although this could be interpreted to mean that not appear to be equivalent. Brca1 and Brca2 mutations have opposite effects in Nuclear localization signals in human BRCA2 have annealing-type repair events, this conclusion is tenuous, been mapped to the region of the protein that is because Rad51-mediated repair can also give rise to deleted in the mouse Brca2lex1 and Brca2lex2 alleles deletion products at a repeat, making it difficult to (Spain et al., 1999; Yano et al., 2000). Cell fractiona- tease apart the contributions of the various pathways. tion studies, however, indicated that the truncated Human BRCA2 has also been implicated in mouse Brca2 peptide in the Brca2lex1/lex2 cells still homologous recombination through the examination localizes appropriately to the nucleus (Moynahan et of a tumor-derived cell line. The human pancreatic al., 2001b). Consistent with this, another study has adenocarcinoma cell line CAPAN-1 carries a single found that the N-terminus of Brca2 is sufficient for mutated BRCA2 allele which encodes a peptide that is nuclear localization (Sarkisian et al., 2001). The Brca2 truncated within BRC7 (Goggins et al., 1996). peptide in the Brca1lex1/lex2 cells was also found to Homologous repair of a double-strand break was interact with Rad51 (Moynahan et al., 2001b), examined in CAPAN-1 cells and found to be severely presumably through the BRC repeat region which is compromised, with an estimated approximately 100- still intact in the truncated peptide. Thus, the C- fold reduced level compared with other cell lines terminus of Brca2 has a role in homologous (Moynahan et al., 2001b). Gene targeting was also recombination that goes beyond proper cellular found to be reduced in the CAPAN-1 cells relative to localization of the protein or simply providing the cells in which wild-type BRCA2 has been re-expressed interaction site for Rad51. Although a human BRCA2 (Xia et al., 2001). Consistent with homologous mutant tumor cell line has also been found to be recombination defects, DNA damage-induced RAD51 defective for homologous recombination (see below), it focus formation is impaired in the CAPAN-1 cell line is not clear if these results with mouse Brca2 are (Yuan et al., 1999), as in the Brca2Tr/DEx27 cell line strictly applicable in detail to human BRCA2. In (Tutt et al., 2001) and a hamster Brca2-deficient addition to a dependence of the human protein on the hamster cell line (Kraakman-van der Zwet et al., C-terminus for nuclear localization, the human protein 2002). Interestingly, localization of RAD51 to the differs from the mouse protein in that there is no nucleus has also been shown to be altered in the evidence that the C-terminus of human BRCA2 CAPAN-1 (Davies et al., 2001) and hamster cell lines interacts with RAD51 (Chen et al., 1998b). (Kraakman-van der Zwet et al., 2002). A second Brca2 mutant mouse embryonic stem cell These studies in BRCA2 mutant mouse and human line has been reported to be impaired for homologous cell lines have been complemented by those using a recombination (Tutt et al., 2001). This mutant has two dominant negative approach in which BRC repeat different Brca2 hypomorphic alleles by gene targeting: peptides are expressed in otherwise BRCA2 wild-type the DEx27 allele, which like lex1 is detected for the cell lines. The rationale for this approach is that the terminal exon 27, and the Tr allele, in which Brca2 is BRC repeat peptides will not have the full biochemical truncated at amino acid 2014 at BRC7. Peptides from activity of intact BRCA2 but will still interact with both alleles were shown to interact with Rad51 by co- RAD51 and compromise the interaction of functional immunoprecipitation, although Rad51 nuclear focus wild-type BRCA2 with RAD51. Consistent with this, formation upon DNA damage is impaired in the expression of a single BRC repeat fused to a Brca2Tr/DEx27 cell line (Tutt et al., 2001). When tested heterologous protein reduces the co-immunoprecipita-

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8989 tion of BRCA2 and RAD51 (Chen et al., 1999) and to determine the effect of a BRC repeat peptide on the results in inpaired RAD51 focus formation upon DNA ability of RAD51 to bind DNA (Davies et al., 2001). damage (Chen et al., 1999; Yuan et al., 1999). When RAD51 was preincubated with an excess of Functional significance to these results with regards BRC repeat peptide, the ability of RAD51 to bind to homologous recombination assays is seen in that DNA was severely compromised and nucleoprotein BRC repeat expression diminishes both BRCA2- filament formation was disrupted. Gel filtration studies dependent gene targeting (Xia et al., 2001) and indicate that RAD51 self-associates to form a number homologous repair of a double-strand break (Stark et of species, i.e., aggregates, rings, and monomers, but al., 2002). that the addition of a BRC repeat peptide disrupts the BRCA2, like BRCA1, colocalizes with RAD51 ability to self-associate, such that RAD51 runs as a during meiosis on chromosome axes prior to homolog monomer associated with the BRC repeat peptide synapsis (Chen et al., 1998a). Thus far, however, (Davies et al., 2001). These results are consistent with hypomorphic mouse lines have not been informative those obtained in vivo, in which expression of a single about a role in meiotic recombination. Males from the BRC repeat impairs homologous recombination (Stark Brca2Tr/Tr line had an absence of germ cells in the et al., 2002; Xia et al., 2001), as well as with the recent testis, and females were devoid of obvious follicles, structure determination of a BRC peptide bound to suggesting a loss of germ cells prior to meiosis in one RAD51 (Pellegrini et al., 2002). or both sexes (Connor et al., 1997). By contrast, mice Strand exchange reactions of RAD51 involve a in which only exon 27 was deleted were fertile for both single-stranded DNA molecule and a homologous sexes (McAllister et al., 2002). DNA duplex. This reaction is strongly dependent on Ustilago maydis provides a complementary, geneti- the single-stranded binding RPA, presumably to cally tractable system in which to examine the function remove secondary structure from single-stranded of a BRCA2 ortholog in DNA repair (Kojic et al., DNA to make it accessible for base pairing (Baumann 2002). BRH2 was disrupted to create a null allele and and West, 1997). The structural similarity of three then the brh2 strain was examined for recombination domains of the BRCA2 C-terminal domain with the defects. Homologous recombination was found to be oligonucleotide binding folds of RPA led Yang et al. as severely reduced in the brh2 strain of Ustilago as in (2002) to determine whether the BRCA2 C-terminal the rad51 mutant strain. For example, homologous domain, in complex with DSS1, could affect the repair of a plasmid double-strand break from an intact requirement for RPA in RAD51-mediated strand chromosome is reduced approximately 100-fold in both exchange reactions. Although the BRCA2 C-terminal the brh2 and rad51 strains. Recombination between domain could not substitute for RPA, this domain chromosome homologs is also reduced, in this case could enhance strand exchange in the presence of RPA. greater than 200-fold for spontaneous events and 2000- Normally, this reaction has a defined order of addition, fold for UV-induced events. BRH2, like RAD51, is with RAD51 and DNA incubated together first and absolutely required for a productive meiosis in RPA added second, presumably because RPA addition Ustilago, strongly implicating it in meiotic recombina- first will block access of RAD51 to single-stranded tion in this organism. Similar to the observation of DNA. Accessory factors, which have been termed chromosome abnormalities in mammalian BRCA1 and mediators, have been identified which overcome this BRCA2 mutants, genomic instability was elevated in inhibition (Gasior et al., 2001). The BRCA2 C-terminal the brh2 mutant, as evidenced by a 10-fold increase in was found to have such an activity, allowing the loss of heterozygosity in diploids, as determined simultaneous addition of RPA, RAD51, and DNA for genetically, and by an increased frequency of aberrant efficient strand exchange (Yang et al., 2002). chromosomes, as determined by pulsed field gel The finding that the BRCA2 C-terminal domain electrophoresis. Thus, mutation of BRH2 in Ustilago stimulates RAD51 strand exchange could readily recapitulates phenotypes of mammalian BRCA2 account for the impaired homologous recombination mutants, indicating that this fungal system holds found in BRCA2 mutants. Notably, this region is not significant promise to gain further insight into cellular present in the truncated BRCA2 peptide in CAPAN-1 functions of BRCA2. cells and in some of the mouse Brca2 truncation alleles, e.g., Brca2Tr2014. The C-terminal domain used in the in vitro studies is predicted to be intact, however, in other Biochemical studies of BRCA2 – an accessory factor for mouse alleles, i.e., Brca2lex1 and Brca2DEx27. Never- RAD51-mediated strand exchange? theless, the function of the C-terminal domain could possibly be perturbed by the deletion of the exon 27 Two recent studies have begun to examine the role of sequences. BRCA2 protein in vitro on RAD51 protein. RAD51 promotes strand exchange between homologous DNA molecules, a reaction thought to be central to Nonhomologous end-joining (NHEJ) of double-strand homologous recombination reactions (Baumann et al., breaks and BRCA1 1996). As part of this reaction, RAD51 binds DNA to form nucleoprotein filaments. The recognition that the In addition to RAD51, BRCA1 has also been found in BRC repeats of BRCA2 bound RAD51 led to a study nuclear foci with a number of other proteins involved

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8990 in the DNA damage response (Wang et al., 2000), mouse embryonic fibroblasts, the level of NHEJ to including the RAD50 complex (Zhong et al., 1999). produce multimeric forms was reduced 3 to 10-fold Like RAD51, RAD50 is involved in homologous relative to extracts from control cells at low Mg2+ recombination, but unlike RAD51, RAD50 is also concentrations, although the level of NHEJ at high implicated in double-strand break repair by NHEJ Mg2+ concentrations was not affected (Zhong et al., (Haber, 1998; Paull, 2001). NHEJ of DNA ends can be 2002a). In this study, human cell extracts immuno- a precise form of DNA repair if the ends are depleted for BRCA1 were also found to have a reduced undamaged; however, if base damage occurs or level of NHEJ, as were extracts depleted for RAD50. nucleotides are otherwise lost or damaged, mammalian In another study, cell extracts from three BRCA1+/7 cells can nevertheless rejoin the ends, but the rejoining lymphoblastoid lines from carriers prepared according is imprecise (mutagenic) in relation to the original to Baumann and West (1998) were examined for NHEJ sequence present prior to DNA damage. Thus, relative (Baldeyron et al., 2002). The extracts from the to homologous recombination, NHEJ is an imprecise BRCA1+/7 mutant cells were found to support less mode of repair. plasmid multimerization than control BRCA1+/+ cell NHEJ has recently been examined in BRCA1 lines. The BRCA1+/7 lymphoblastoid cell lines, which mutants by several groups, with complex results. In each carried a different BRCA1 mutation, had a lower the mouse Brca1D223-763/D223-763 mutant, imprecise than expected level of wild-type BRCA1 protein NHEJ was examined after a double-strand break was considering the heterozygosity of the lines, possibly introduced in vivo at a defined chromosomal locus accounting for the reduced NHEJ in these lines. This (Moynahan et al., 1999). Unlike homologous repair, study also used in vivo plasmid-based rejoining assays to the level of NHEJ was not reduced in this line, but examine NHEJ in the BRCA1+/7 lymphoblastoid lines rather was found to be slightly elevated. NHEJ was (Baldeyron et al., 2002). In this type of assay, a examined more recently using a similar approach in a linearized plasmid is introduced into cells, and then second mouse cell line, a unique embryonic fibroblast the plasmid DNA is recovered and transformed into line derived from mice carrying an apparently null bacteria. Using this plasmid based approach, the allele of Brca1, as well as mutant p53 (Zhong et al., BRCA1+/7 lymphoblastoid lines did not show a major 2002b). As with the Brca1D223-763/D223-763 mutant, the defect, i.e., greater than 10-fold, in the level of overall level of imprecise NHEJ in these Brca17/7 recircularization of the plasmid. However, the NHEJ p537/7 cells was not noticeably reduced relative to products showed a decreased fraction of precise ligation p537/7 control cells. However, a specific NHEJ events (i.e., on average sixfold lower), indicating a product involving a 4-bp microhomology was reduced greater propensity to sequence alterations at the DNA 450-fold, suggesting that Brca1 may influence a subset ends prior to rejoining. The differential results obtained of NHEJ events. Although intriguing, NHEJ pathways in the level of rejoining between the cell extract and in have not been sufficiently characterized in mammalian vivo studies may possibly reflect that the in vivo assay cells to understand the significance of the loss of this measures both precise and imprecise ligation events, particular product. whereas the cell extract assay primarily measures precise NHEJ is key to the repair of double-strand breaks ligation events. generated during V(D)J recombination; thus, successful A third study has used cell free extracts prepared antigen receptor rearrangement in the immune system according to Feldmann et al. (2000) from a human is indicative of functional NHEJ pathways. A condi- BRCA1 mutant cell line that expresses no wild-type tional Brca1 null allele has been constructed to protein (i.e., HCC1937 breast carcinoma) (Me´ rel et al., examine the role of Brca1 in T cell development 2002). Extracts from the HCC1937 cell line were found (Mak et al., 2000). When this allele is conditionally in this study to support similar levels and accuracy of inactivated, exons 5 and 6 are deleted to result in a NHEJ as extracts from control cell lines (Me´ rel et al., transcript encoding only the first 49 amino acids of 2002). The similar level of rejoining obtained in this Brca1. Although thymocyte development was arrested study is consistent with the normal in vivo NHEJ in a p53-dependent manner in these mice, apparently kinetics of ionizing radiation-induced double-strand due to chromosome abnormalities, T cell receptor breaks that have been reported for this cell line (Wang rearrangement was not obviously impaired, indicating et al., 2001). that NHEJ was functional during V(D)J recombination Thus, a few studies indicate a role for BRCA1 in in the absence of (Mak et al., 2000). certain types of NHEJ, while others do not. As each of Cell free extracts have also been used to examine the these studies utilize different assay systems and cell involvement of BRCA1 in NHEJ. In two variations of lines with different mutations, it is as yet difficult to these in vitro assays, monomer length plasmid can be decipher whether deficiencies in particular pathways of converted to dimers, trimers, and higher multimeric NHEJ are a general outcome of BRCA1 mutation. forms, mostly by precise ligation of DNA ends A few reports have examined the BRCA2 mutant (Baumann and West, 1998) or, with a different cell CAPAN-1 cell line for NHEJ, using cell extracts extraction procedure, also recircularized, by either (Me´ rel et al., 2002; Yu et al., 2000) or rejoining of precise ligation or imprecise rejoining (Feldmann et radiation-induced double-strand breaks (Wang et al., al., 2000). In cell extracts prepared according to 2001). Each of these reports reached the same Baumann and West (1998) from the Brca17/7 p537/7 conclusion, i.e., that NHEJ is not affected in this line.

Oncogene BRCA1, BRCA2, and homologous DNA repair M Jasin 8991 Checkpoint defects and BRCA1 or BRCA2 deﬁciency Conclusions

DNA repair deficiencies have the potential to make The association of BRCA1 and BRCA2 with RAD51 cells hypermutable. However, if unrepaired damage implicated these proteins in homologous recombina- triggers a cell cycle checkpoint or apoptosis, repair tion. Recent studies have now given strong support to deficient cells can be eliminated from the population, this, in particular the finding of homologous recombi- such that mutations cannot efficiently accumulate. nation defects in BRCA2 mutant cell lines and a Thus, a checkpoint defect may allow a repair BRCA1 mutant cell line. Furthermore, biochemical deficient phenotype to manifest itself. BRCA1 and structural studies of the BRCA2 C-terminal mutant human cells and Brca2 deficient hamster domain place BRCA2 directly in the recombination cells have been shown to have a defective S phase pathway. Several challenges still remain in terms of checkpoint response to ionizing radiation (Kraak- directly linking recombination defects to tumorigenesis, man-van der Zwet et al., 2002; Xu et al., 2001a), particularly in a manner that explains the tissue suggesting that mutant cells may continue to distribution of the tumors seen in BRCA carriers, progress through the cell cycle with unrepaired and parsing the role of the BRCA proteins in other damage arising during replication. Interestingly, a DNA repair pathways and in checkpoint responses, separation of function mutation in BRCA1 has been especially as regards BRCA1. Certainly, the next few identified in which the ionizing radiation-induced years promise to yield a plethora of other studies which checkpoint has been abrogated, but cell survival will address these issues. Stay tuned. after irradiation is not affected (Turner et al., 2002). Evidence also supports a G2/M checkpoint defect (Chen et al., 1999; Xu et al., 1999, 2001a). Thus, it Acknowledgements is possible that DNA repair and checkpoint defects We thank Richard Baer and Nicole Christ for comments cooperate in BRCA deficient cells to produce the on the manuscript and Larry Norton for support through observed chromosome abnormalities found in these the Wolfson Family Foundation, the Breast Cancer cells. Research Fund, and NIH P01CA94060.

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