**1. Introduction**

326 DNA Repair

Zini, A., Boman, J.M., Belzile, E., and Ciampi, A. (2008). Sperm DNA damage is associated

Zühlke, C., Rless, O., Bockel, B., Lange, H., and Thies, U. (1993). Mitotic stability and meiotic

meta-analysis. Human Reproduction *23*, 2663-2668.

Genetics *2*, 2063-2067.

with an increased risk of pregnancy loss after IVF and ICSI: systematic review and

variability of the (CAG) nrepeat in the Huntington disease gene. Human Molecular

The *BRCA2* gene was the second gene discovered to be associated with early-onset, familial breast cancer. The BRCA2 protein is expressed in breast, ovarian, prostate, and pancreatic tissues and is associated with cancer predisposition in all four, with breast cancer being the most predominant (Goggins, Schutte et al. 1996). BRCA2 is functionally defined as a tumor suppressor and is most critical in maintenance of genomic integrity and DNA repair fidelity. The importance of BRCA2 in maintaining genomic integrity is based on its function to specifically repair double-strand DNA breaks (DSBs) via the process of homologous recombination (HR). However, BRCA2 resolves genomic lesions in concert with a number of DNA repair proteins, the most significant being RAD51 (Sharan, Morimatsu et al. 1997),(Yuan, Lee et al. 1999). RAD51 is a recombinase that is highly conserved, having homologues in *E. coli* and yeast, as well as in mammals. BRCA2 modulates the activity of RAD51 during DNA repair, and they both are found in nuclear DNA damage-induced foci, which are complexes of DNA repair proteins bound to DNA during the process of repair (Roth, Porter et al. 1985; Roth and Wilson 1986; Derbyshire, Epstein et al. 1994; Jackson and Jeggo 1995; Takata, Sasaki et al. 1998; Johnson and Jasin 2000). The relationship between BRCA2 and RAD51 has been determined to be a fundamental interaction in the repair of DSBs.

The role of BRCA2 as a tumor suppressor has been established by its importance in maintaining genomic integrity. The inability of the cell to repair DSBs can potentially cause small-scale lesions in regions of the DNA that encode single genes and incite large-scale lesions, such as chromosomal anomalies. The consequence of such damage can disrupt the normal expression of gene products that are required to regulate cell growth and arrest and induce apoptosis, thereby establishing a cellular environment that can foster malignant transformation.

Cancer cells that express mutated BRCA2 have been shown to have elevated sensitivity to the anti-cancer therapeutics called PARP (Poly [ADP-ribose] polymerase) inhibitors. PARP inhibitors prevent the binding of PARP to sites of damaged DNA, which serves as a signal to initiate DNA repair (Schreiber, Dantzer et al. 2006); (Ratnam and Low 2007). The effectiveness of PARP inhibitors in BRCA2-mutated cells is based on the premise of synthetic lethality, which is when two pathway defects alone are innocuous, but combined become lethal (Ratnam and Low 2007). The unresolved DSBs of BRCA2-mutated cells combined with the inhibition of PARP activity are effective in promoting DNA damage-

*BRCA2* Mutations and Consequences for DNA Repair 329

repeats 1–4, 7, and 8 all interact with RAD51, with repeats 3 and 4 having the strongest affinity (Wong, Pero et al. 1997) (Bignell, Micklem et al. 1997). The second RAD51 binding site is located on the CTD (C-terminal domain) of BRCA2. This RAD51 binding site is described as playing a major role in the regulation of RAD51 recombination activity by displacing the single-strand DNA binding protein replication protein A (RPA) from the exonucleolytically processed 3′-single-strand overhangs of the DSBs, thus allowing RAD51 to bind and form nucleoprotein filaments (Yang, Jeffrey et al. 2002). The CTD portion of BRCA2 has been shown to be highly active in HR-mediated repair with RAD51 (Yang, Jeffrey et al. 2002). This region consists of five domains significant in DNA repair. The first is the α-helical domain, which interacts with the DMC1 protein—a meiosis specific paralog of RAD51 that forms nucleoprotein filaments and catalyzes strand exchange, and that BRCA2 requires for meiotic recombination (Thorslund, Esashi et al. 2007); (Jensen, Carreira et al.). The next three domains are the oligonucleotide–oligosaccharide binding domains (OB1, OB2, OB3) that have structural similarities with ssDNA binding proteins such as replication protein A (RPA). And, the fifth domain is the tower domain, which extends from OB2, and has structural similarities with the DNA binding domains of bacterial site-specific recombinases able to bind double-strand DNA

Fig. 1. Model of homologous recombination (HR)-mediated repair. After a DSB has occurred, the MRN-CtIP complex resects the 5'ends of the break. The 3'ssDNA overhangs are coated with replication protein A (RPA), which is displaced by RAD51. The BRCA1- PALB2-BRCA2 complex facilitates binding of RAD51 to form nucleoprotein filaments which invade the homologous strand, resulting in the D loop intermediate. This is followed by

formation of the Holliday junction and resolution of the DSB.

(Yang, Jeffrey et al. 2002).

induced apoptosis. This finding has established mutated BRCA2 as a potential target in improving present anti-cancer therapeutic regimens.

The information that follows will provide a comprehensive understanding of BRCA2, starting from its functions at the molecular level in maintaining genomic integrity, to describing how deregulation can lead to disease predisposition and development, and concluding with the development of PARP inhibitors that use the DNA repair defects of BRCA2-mutations to improve the sensitivity of anti-cancer treatments towards BRCA2 tumors.

### **2. The role of BRCA2 in DNA repair**

The BRCA2 protein specifically repairs double-strand DNA breaks (DSBs) via the process of homologous recombination (HR), thereby establishing its importance in maintaining genomic integrity. The *BRCA2* gene is found on chromosome 13q12.3 and encodes a protein of 3,418 amino acids, resulting in a molecular weight of approximately 340 kDa. BRCA2 resolves genomic lesions in a complex with several additional DNA repair proteins, the most significant being RAD51 (Sharan, Morimatsu et al. 1997; Yuan, Lee et al. 1999). RAD51 is a highly conserved recombinase, having homologues in *E. coli* and yeast, as well as in mammals. BRCA2 modulates the activity of RAD51 during DNA repair and this relationship is determined to be a fundamental interaction in repair of DSBs.

#### **2.1 The interaction between BRCA2 and the RAD51 recombinase**

RAD51 catalyzes the strand exchange of DNA homologues to promote gene conversion and repair DSBs by HR (Ogawa, Yu et al. 1993) (Benson, Stasiak et al. 1994). HR is one of two pathways of repair of DSBs in mammals—the other being nonhomologous end-joining (NHEJ) (Derbyshire, Epstein et al. 1994), (Jackson and Jeggo 1995), (Roth, Porter et al. 1985), (Roth and Wilson 1986), (Takata, Sasaki et al. 1998) and (Johnson and Jasin 2000), (Figure 1). HR requires the damaged DNA molecule to use the undamaged homologue as a template in order to repair the DSB. NHEJ involves ligation of the DNA ends at the breakpoint junction regardless of whether the original genetic information is still present. As a result, HR confers greater accuracy in repair than NHEJ (Derbyshire, Epstein et al. 1994), (Jackson and Jeggo 1995), (Roth, Porter et al. 1985), (Roth and Wilson 1986), (Takata, Sasaki et al. 1998) and (Johnson and Jasin 2000). Studies performed in mice in which the *Rad51* gene was either mutated or completely knocked out have shown its importance in genomic stability and cell viability (Taki, Ohnishi et al. 1996) and (Sonoda, Sasaki et al. 1998). Nonfunctional RAD51 does not repair chromosome breaks and other DNA lesions, thereby leading to an accumulation of DSBs and stalled replication forks (Taki, Ohnishi et al. 1996) and (Sonoda, Sasaki et al. 1998). In addition, inactivation of the *RAD51* gene causes embryonic lethality (Tsuzuki, Fujii et al. 1996).

#### **2.2 The structure of BRCA2**

Yeast two-hybrid screening assays were used in the discovery of the interaction between RAD51 and BRCA2 (Mizuta, LaSalle et al. 1997), (Wong, Pero et al. 1997), (Chen, Chen et al. 1998) and (Marmorstein, Ouchi et al. 1998). And, studies examining the interaction between the two proteins have collectively shown that BRCA2 has two regions for RAD51 binding. The first region is in the mid-portion of BRCA2 and consists of eight highly conserved amino acid motifs called BRC repeats (Figure 2). The repeats have different binding affinities for RAD51:

induced apoptosis. This finding has established mutated BRCA2 as a potential target in

The information that follows will provide a comprehensive understanding of BRCA2, starting from its functions at the molecular level in maintaining genomic integrity, to describing how deregulation can lead to disease predisposition and development, and concluding with the development of PARP inhibitors that use the DNA repair defects of BRCA2-mutations to improve the sensitivity of anti-cancer treatments towards BRCA2-

The BRCA2 protein specifically repairs double-strand DNA breaks (DSBs) via the process of homologous recombination (HR), thereby establishing its importance in maintaining genomic integrity. The *BRCA2* gene is found on chromosome 13q12.3 and encodes a protein of 3,418 amino acids, resulting in a molecular weight of approximately 340 kDa. BRCA2 resolves genomic lesions in a complex with several additional DNA repair proteins, the most significant being RAD51 (Sharan, Morimatsu et al. 1997; Yuan, Lee et al. 1999). RAD51 is a highly conserved recombinase, having homologues in *E. coli* and yeast, as well as in mammals. BRCA2 modulates the activity of RAD51 during DNA repair and this

RAD51 catalyzes the strand exchange of DNA homologues to promote gene conversion and repair DSBs by HR (Ogawa, Yu et al. 1993) (Benson, Stasiak et al. 1994). HR is one of two pathways of repair of DSBs in mammals—the other being nonhomologous end-joining (NHEJ) (Derbyshire, Epstein et al. 1994), (Jackson and Jeggo 1995), (Roth, Porter et al. 1985), (Roth and Wilson 1986), (Takata, Sasaki et al. 1998) and (Johnson and Jasin 2000), (Figure 1). HR requires the damaged DNA molecule to use the undamaged homologue as a template in order to repair the DSB. NHEJ involves ligation of the DNA ends at the breakpoint junction regardless of whether the original genetic information is still present. As a result, HR confers greater accuracy in repair than NHEJ (Derbyshire, Epstein et al. 1994), (Jackson and Jeggo 1995), (Roth, Porter et al. 1985), (Roth and Wilson 1986), (Takata, Sasaki et al. 1998) and (Johnson and Jasin 2000). Studies performed in mice in which the *Rad51* gene was either mutated or completely knocked out have shown its importance in genomic stability and cell viability (Taki, Ohnishi et al. 1996) and (Sonoda, Sasaki et al. 1998). Nonfunctional RAD51 does not repair chromosome breaks and other DNA lesions, thereby leading to an accumulation of DSBs and stalled replication forks (Taki, Ohnishi et al. 1996) and (Sonoda, Sasaki et al. 1998). In addition, inactivation of the *RAD51* gene causes embryonic lethality

Yeast two-hybrid screening assays were used in the discovery of the interaction between RAD51 and BRCA2 (Mizuta, LaSalle et al. 1997), (Wong, Pero et al. 1997), (Chen, Chen et al. 1998) and (Marmorstein, Ouchi et al. 1998). And, studies examining the interaction between the two proteins have collectively shown that BRCA2 has two regions for RAD51 binding. The first region is in the mid-portion of BRCA2 and consists of eight highly conserved amino acid motifs called BRC repeats (Figure 2). The repeats have different binding affinities for RAD51:

relationship is determined to be a fundamental interaction in repair of DSBs.

**2.1 The interaction between BRCA2 and the RAD51 recombinase** 

improving present anti-cancer therapeutic regimens.

**2. The role of BRCA2 in DNA repair** 

(Tsuzuki, Fujii et al. 1996).

**2.2 The structure of BRCA2** 

tumors.

repeats 1–4, 7, and 8 all interact with RAD51, with repeats 3 and 4 having the strongest affinity (Wong, Pero et al. 1997) (Bignell, Micklem et al. 1997). The second RAD51 binding site is located on the CTD (C-terminal domain) of BRCA2. This RAD51 binding site is described as playing a major role in the regulation of RAD51 recombination activity by displacing the single-strand DNA binding protein replication protein A (RPA) from the exonucleolytically processed 3′-single-strand overhangs of the DSBs, thus allowing RAD51 to bind and form nucleoprotein filaments (Yang, Jeffrey et al. 2002). The CTD portion of BRCA2 has been shown to be highly active in HR-mediated repair with RAD51 (Yang, Jeffrey et al. 2002). This region consists of five domains significant in DNA repair. The first is the α-helical domain, which interacts with the DMC1 protein—a meiosis specific paralog of RAD51 that forms nucleoprotein filaments and catalyzes strand exchange, and that BRCA2 requires for meiotic recombination (Thorslund, Esashi et al. 2007); (Jensen, Carreira et al.). The next three domains are the oligonucleotide–oligosaccharide binding domains (OB1, OB2, OB3) that have structural similarities with ssDNA binding proteins such as replication protein A (RPA). And, the fifth domain is the tower domain, which extends from OB2, and has structural similarities with the DNA binding domains of bacterial site-specific recombinases able to bind double-strand DNA (Yang, Jeffrey et al. 2002).

Fig. 1. Model of homologous recombination (HR)-mediated repair. After a DSB has occurred, the MRN-CtIP complex resects the 5'ends of the break. The 3'ssDNA overhangs are coated with replication protein A (RPA), which is displaced by RAD51. The BRCA1- PALB2-BRCA2 complex facilitates binding of RAD51 to form nucleoprotein filaments which invade the homologous strand, resulting in the D loop intermediate. This is followed by formation of the Holliday junction and resolution of the DSB.

*BRCA2* Mutations and Consequences for DNA Repair 331

filaments which invade the homologous template and form a Holliday junction. In the absence of PALB2, cells exhibit genomic instability and treatment with drugs that cause

*BRCA2* and its predecessor, *BRCA1*, were the first genes to be discovered that were associated with early-onset, familial breast cancer. Furthermore, germline mutations of *BRCA2* are also responsible for hereditary forms of ovarian, prostate and pancreatic cancer; however, the risk of acquiring breast cancer is most prevalent. Moreover, the risk for breast cancer is 50-80%, however, the degree of penetrance has been shown to vary (Tonin, Weber

Most mutations in BRCA2 are the result of small deletions and insertions. In fact, a *BRCA2* mutation that has been of interest for almost two decades is the 6174delT mutation, in which the thymine at position 6174 is deleted. This mutation disrupts BRC repeats 5 and 6, and introduces a premature stop codon that abruptly truncates the protein (Neuhausen, Gilewski et al. 1996; Oddoux, Struewing et al. 1996; Roa, Boyd et al. 1996; Abeliovich, Kaduri et al. 1997; Levy-Lahad, Catane et al. 1997)**.** The truncated form no longer possesses the CTD region, which comprises the domains required for DNA repair and recombination, the second RAD51 binding site, TR2/S3291, and the putative nuclear localization signals. As a consequence, cells with this mutation exhibit inefficient repair of DSBs, loss of genomic stability, and sensitivity to radiation and DNA crosslinking agents (Goggins, Schutte et al. 1996),(Ozcelik, Schmocker et al. 1997). The 6174delT is a founder mutation in the Ashkenazi Jewish population at a frequency of 1.36% ((Tonin, Weber et al. 1996)). And, it is the only BRCA2 mutation, along with three BRCA1 mutations, that is carried in 78-96% of Ashkenazi Jews with detectable mutations (Oddoux, Guillen-Navarro

Another *BRCA2* mutation that was also discovered to have a founder's effect is the 999del5 mutation, which was discovered in an Icelandic population (Thorlacius, Olafsdottir et al. 1996). It is a five base-pair deletion that starts at nucleotide 999, codon 257 in exon 9. The mutation introduces a frame-shift that prematurely truncates the protein, and renders it nonfunctional, similar to the effect of the 6174delT founder mutation in the Ashkenazi Jewish population. Carriers of the mutation exhibit familial forms of male or female breast, prostate or pancreatic cancer. However, there are varying forms of penetrance, in which some carriers have never been diagnosed with cancer. In fact, there is either absolutely no phenotypic expression or

In a study of BRCA1/2 mutations performed in a Serbian population, one family was shown to carry a BRCA2 mutation that was an insertion of two nucleotides, c.4367\_4368dupTT (Dobricic, Brankovic-Magic et al.). The mutation causes a frame-shift that alters codons 1381- 1387 and introduces a premature stop codon at position 1388, resulting in a loss of > 2,000 amino acids at the C-terminus. The protein product lacks BRC repeats 3-8, as well as the crucial CTD and TR2 domains, rendering BRCA2 completely non-functional in regulating RAD51 activity, as well as in promoting HR-mediated repair of DSBs (Dobricic, Brankovic-

diagnosis of varying forms of cancer (Thorlacius, Olafsdottir et al. 1996).

**3. Mutations of BRCA2, DNA repair fidelity and disease predisposition** 

inter-strand crosslinks leads to increased chromosome breakage.

et al. 1996).

Magic et al.)

**3.1 Mutated BRCA2 in familial cancers** 

et al. 1999) (Mangold, Wang et al.)

Also located on the C-terminus of BRCA2 are its two nuclear localization signals (NLSs) (Spain, Larson et al. 1999) and (Yano, Morotomi et al. 2000). As a result, C-terminal mutations which disrupt, or truncations which remove, the NLSs are extremely detrimental to BRCA2 DNA repair functions, because they prevent nuclear localization. And, cell lines that have nonfunctional or absent BRCA2 NLSs primarily exhibit cytoplasmic localization of RAD51 after induction of DSBs by ionizing radiation (IR) (Spain, Larson et al. 1999). BRCA2 also interacts with RAD51 via a separate motif located at its C-terminus (Esashi, Christ et al. 2005). This interaction is regulated by cell cycle (CDK)-dependent phosphorylation of serine 3291 in exon 27 (and has been referred to, in some instances, as the "TR2" domain) and appears to function as a "switch" controlling recombinational repair activity during the transition from S/G2 to M phase in the cell cycle (Esashi, Christ et al. 2005). This phosphorylation site appears to be crucial in the checkpoint control mechanisms involved in the DNA repair pathway involving BRCA2.

Fig. 2. Schematic of BRCA2. Starting at the N-terminus, the PALB2, the DMC1 and the two RAD51 binding sites on BRCA2 are indicated by black bars. The mid-portion contains eight highly-conserved BRC repeats. The CTD contains the α-helical domain, three OB-folds, the TR2 (location of S3291) domain, and putative nuclear localization signals (NLSs). The tower domain (not shown) extends from the second OB-fold (OB2).

The N-terminal region of BRCA2 does not bind RAD51; however, it does interact with a protein that is equally crucial to maintenance of genomic integrity, which is PALB2 (partner and localizer of BRCA2) (Xia, Sheng et al. 2006); (Rahman, Seal et al. 2007). PALB2 has been observed complexed with DNA damage-induced BRCA1/BRCA2 nuclear foci (Sy, Huen et al. 2009), (Zhang, Fan et al. 2009; Zhang, Ma et al. 2009). Subsequent studies have shown that PALB2 is crucial in the localization of BRCA2 to sites of DNA damage via associations with chromatin structures and in HR-mediated DNA repair ((Sy, Huen et al. 2009), (Zhang, Fan et al. 2009; Zhang, Ma et al. 2009). This indirectly influences the localization of RAD51 to sites of DNA damage, due to its reliance on BRCA2. During the process of HR-mediated repair, PALB2 appears to be crucial in "D-loop" formation, (Buisson, Dion-Cote et al.)2010) (figure 2). This is the step in which the 3' overhangs of the dsDNA break, resulting from resection of the 5' ends of the break, are coated with RAD51 protein to form nucleoprotein

Also located on the C-terminus of BRCA2 are its two nuclear localization signals (NLSs) (Spain, Larson et al. 1999) and (Yano, Morotomi et al. 2000). As a result, C-terminal mutations which disrupt, or truncations which remove, the NLSs are extremely detrimental to BRCA2 DNA repair functions, because they prevent nuclear localization. And, cell lines that have nonfunctional or absent BRCA2 NLSs primarily exhibit cytoplasmic localization of RAD51 after induction of DSBs by ionizing radiation (IR) (Spain, Larson et al. 1999). BRCA2 also interacts with RAD51 via a separate motif located at its C-terminus (Esashi, Christ et al. 2005). This interaction is regulated by cell cycle (CDK)-dependent phosphorylation of serine 3291 in exon 27 (and has been referred to, in some instances, as the "TR2" domain) and appears to function as a "switch" controlling recombinational repair activity during the transition from S/G2 to M phase in the cell cycle (Esashi, Christ et al. 2005). This phosphorylation site appears to be crucial in the checkpoint control mechanisms involved in

Fig. 2. Schematic of BRCA2. Starting at the N-terminus, the PALB2, the DMC1 and the two RAD51 binding sites on BRCA2 are indicated by black bars. The mid-portion contains eight highly-conserved BRC repeats. The CTD contains the α-helical domain, three OB-folds, the TR2 (location of S3291) domain, and putative nuclear localization signals (NLSs). The tower

The N-terminal region of BRCA2 does not bind RAD51; however, it does interact with a protein that is equally crucial to maintenance of genomic integrity, which is PALB2 (partner and localizer of BRCA2) (Xia, Sheng et al. 2006); (Rahman, Seal et al. 2007). PALB2 has been observed complexed with DNA damage-induced BRCA1/BRCA2 nuclear foci (Sy, Huen et al. 2009), (Zhang, Fan et al. 2009; Zhang, Ma et al. 2009). Subsequent studies have shown that PALB2 is crucial in the localization of BRCA2 to sites of DNA damage via associations with chromatin structures and in HR-mediated DNA repair ((Sy, Huen et al. 2009), (Zhang, Fan et al. 2009; Zhang, Ma et al. 2009). This indirectly influences the localization of RAD51 to sites of DNA damage, due to its reliance on BRCA2. During the process of HR-mediated repair, PALB2 appears to be crucial in "D-loop" formation, (Buisson, Dion-Cote et al.)2010) (figure 2). This is the step in which the 3' overhangs of the dsDNA break, resulting from resection of the 5' ends of the break, are coated with RAD51 protein to form nucleoprotein

domain (not shown) extends from the second OB-fold (OB2).

the DNA repair pathway involving BRCA2.

filaments which invade the homologous template and form a Holliday junction. In the absence of PALB2, cells exhibit genomic instability and treatment with drugs that cause inter-strand crosslinks leads to increased chromosome breakage.
