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

*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 et al. 1996).

### **3.1 Mutated BRCA2 in familial cancers**

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

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

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-Magic et al.)

*BRCA2* Mutations and Consequences for DNA Repair 333

a nuclear ubiquitin E3 ligase complex. The complex proceeds to monoubiquitylate the FANCD2 and FANC1 proteins thereby causing them to move to chromatin structures and form nuclear foci at sites of DNA damage. The FA complex interacts with FA members, FANCD1/BRCA2, FANCN/PALB2 and FANCJ, along with other DNA repair proteins to promote HR-mediated resolution of DSBs. Given that this pathway is involved in HRmediated repair, it was not surprising to discover the involvement of RAD51 and BRCA1 downstream in the FA DNA repair pathway. And, because of the involvement of BRCA2, along with BRCA1, this pathway is now referred to as the Fanconi Anemia/BRCA pathway

To activate BRCA2, a sequence of cell signaling events is initiated in response to DNA damage, called the DNA damage response (or DDR). When the cell has experienced DSBs, either by exogenous sources such as ionizing radiation or exposure to crosslinking agents such as cisplatin, or endogenous sources such as free radicals and stalled replication forks, the goal is to immediately arrest cell division and repair the damage. When efficient DNA repair does not occur, apoptosis is induced to prevent propagation of genetic mutations. The phosphoinositide 3-kinase related kinases (PIKKs), ataxia-telangiectasia mutated kinase (ATM), and ATM and Rad 3-related kinase (ATR), are crucial in the detection and subsequent resolution of DNA damage. Furthermore, they are also involved in the Fanconi Anemia pathway, as previously described. ATM and ATR "cross-talk" with each other, given that ATM activates ATR in response to ionizing radiation, and ATR activates ATM in response to ultraviolet light. With respect to the DDR pathway that involves BRCA2, resolution of DSBs is initiated by activation of ATR, after phosphorylation by ATM. ATR proceeds to phosphorylate and active Chk1, which then phosphorylates RAD51. Chk1 arrests the cells in S and G2 phases to ensure DNA is repaired before synthesis and cell division. At this point, RAD51 is now able to engage in HR-mediated repair of DSBs under

BRCA2 appears to play a crucial role during S and G2 phases of the cell cycle. First, during S phase, replication forks can stall and collapse due to exogenous or endogenous sources of damage. A DNA strand break at a replication fork can mimic a DSB as a result of the nascent DNA chain that is being synthesized at the fork. At this point, activated RAD51 is required to repair the break and subsequently stalled fork. It has been proposed that deficient BRCA2, which functions to regulate RAD51 during HR-mediated repair, may be a major cause of diseases resulting from an accumulation of stalled replication forks and consequential DNA breaks that remain unrepaired (Lomonosov, Anand et al. 2003). And, with respect to G2 phase, in a study where the binding of BRCA2 with RAD51 was inhibited in cells expressing the BRC4 repeat, which competed against endogenous full-length BRCA2, there was a failure to initiate radiation-induced G2/M checkpoint arrest. These results implied that the interaction between BRCA2 and RAD51 was imperative for G2/M

The majority of *BRCA2* mutations that are associated with cancer predisposition tend be truncations that remove substantial portions of the CTD, which is where the domains required for DNA repair are located. This region of the protein also appears to be significant in cell cycle changes due to the DNA damage response, via the TR2 domain. The TR2 domain contains a serine at 3291 that is CDK phosphorylated and appears to be one method

**3.3 BRCA2 in cell cycle signaling and the DNA damage response** 

the regulation of BRCA2 (McNeely, Conti et al.), (Connell, Shibata et al.).

checkpoint control (Chen, Chen et al. 1999).

or network (D'Andrea).

#### **3.2 Mutated BRCA2 in the development of Fanconi Anemia**

Another inheritable condition resulting from mutated BRCA2 is the disorder Fanconi Anemia (FA). The disorder is rare and is characterized by aplastic anemia in childhood, susceptibility to leukemia and cancer, and hypersensitivity of FA cells to interstrand crosslinking agents, such as cisplatin (D'Andrea, 2010). The FA proteins are the products of 13 genes that comprise the following subtypes, FA-A, B, C, D1, D2, E, F, G, I, J, L, M, and N. And, eight of those gene products encoding proteins FANCA-C, FANCE-G, FANCL, and FANCM form a nuclear multi-protein core complex (the FA complex) that functions in the DNA repair pathway. Furthermore, it was discovered that genes underlying the FA-D1 (*FANCD1*) and FA-N (*FANCN*) subtypes were *BRCA2* and *PALB2*, respectively. Ultimately, the multi-protein core complex is responsible for monoubiquitylating FANCD2 on lysine 561 in order to activate the Fanconi Anemia pathway in response to S-phase progression or DNA damage (Zhi, Wilson et al. 2009), (Figure 3).

Fig. 3. Schematic of the Fanconi Anemia Pathway. After DNA damage, the ATR kinase phosphorylates and activates the FA core complex, comprised of FANCA, -B, -C, -E, -F, -G, - L, and –M. The complex functions as an E3 ligase and monoubiquitinates the FANCD2/FANCI complex, which then targets chromatin, where it assembles with other DNA repair proteins and FANCD1/BRCA2 and FANCN/PALB2 to repair damaged DNA.

The discovery of the FANCD1 protein being identified as BRCA2 was surprising, yet quite rational given the similarities between FANCD1 and BRCA2 mutated cells. They both exhibit chromosomal instabilities, sensitivity to ionizing radiation and crosslinking agents, and inefficient HR-mediated repair of DSBs. The role of FANCD1/BRCA2 in this protein complex is to act downstream in concert with the FA complex, additional FA members, and DNA repair proteins. However, Fanconi Anemia, subtype D1 is caused by biallelic inactivation of BRCA2; however, risk of breast, ovarian, prostate, and pancreatic cancers are associated with heterozygous BRCA2 mutations (Howlett, Taniguchi et al. 2002).

The FA proteins function in a DNA damage repair pathway, with the multi-protein core complex ultimately being responsible for monoubiquitylating the FANCD2 and FANC1 proteins, in response to DNA damage or entry into S phase of the cell cycle. Activation of the core complex is initiated by phosphorylation of FA proteins by the DNA damage sensing kinases ATM and ATR. After phosphorylation, the core complex assembles to form

Another inheritable condition resulting from mutated BRCA2 is the disorder Fanconi Anemia (FA). The disorder is rare and is characterized by aplastic anemia in childhood, susceptibility to leukemia and cancer, and hypersensitivity of FA cells to interstrand crosslinking agents, such as cisplatin (D'Andrea, 2010). The FA proteins are the products of 13 genes that comprise the following subtypes, FA-A, B, C, D1, D2, E, F, G, I, J, L, M, and N. And, eight of those gene products encoding proteins FANCA-C, FANCE-G, FANCL, and FANCM form a nuclear multi-protein core complex (the FA complex) that functions in the DNA repair pathway. Furthermore, it was discovered that genes underlying the FA-D1 (*FANCD1*) and FA-N (*FANCN*) subtypes were *BRCA2* and *PALB2*, respectively. Ultimately, the multi-protein core complex is responsible for monoubiquitylating FANCD2 on lysine 561 in order to activate the Fanconi Anemia pathway in response to S-phase progression or

Fig. 3. Schematic of the Fanconi Anemia Pathway. After DNA damage, the ATR kinase phosphorylates and activates the FA core complex, comprised of FANCA, -B, -C, -E, -F, -G, -

FANCD2/FANCI complex, which then targets chromatin, where it assembles with other DNA repair proteins and FANCD1/BRCA2 and FANCN/PALB2 to repair damaged DNA. The discovery of the FANCD1 protein being identified as BRCA2 was surprising, yet quite rational given the similarities between FANCD1 and BRCA2 mutated cells. They both exhibit chromosomal instabilities, sensitivity to ionizing radiation and crosslinking agents, and inefficient HR-mediated repair of DSBs. The role of FANCD1/BRCA2 in this protein complex is to act downstream in concert with the FA complex, additional FA members, and DNA repair proteins. However, Fanconi Anemia, subtype D1 is caused by biallelic inactivation of BRCA2; however, risk of breast, ovarian, prostate, and pancreatic cancers are

L, and –M. The complex functions as an E3 ligase and monoubiquitinates the

associated with heterozygous BRCA2 mutations (Howlett, Taniguchi et al. 2002).

The FA proteins function in a DNA damage repair pathway, with the multi-protein core complex ultimately being responsible for monoubiquitylating the FANCD2 and FANC1 proteins, in response to DNA damage or entry into S phase of the cell cycle. Activation of the core complex is initiated by phosphorylation of FA proteins by the DNA damage sensing kinases ATM and ATR. After phosphorylation, the core complex assembles to form

**3.2 Mutated BRCA2 in the development of Fanconi Anemia** 

DNA damage (Zhi, Wilson et al. 2009), (Figure 3).

a nuclear ubiquitin E3 ligase complex. The complex proceeds to monoubiquitylate the FANCD2 and FANC1 proteins thereby causing them to move to chromatin structures and form nuclear foci at sites of DNA damage. The FA complex interacts with FA members, FANCD1/BRCA2, FANCN/PALB2 and FANCJ, along with other DNA repair proteins to promote HR-mediated resolution of DSBs. Given that this pathway is involved in HRmediated repair, it was not surprising to discover the involvement of RAD51 and BRCA1 downstream in the FA DNA repair pathway. And, because of the involvement of BRCA2, along with BRCA1, this pathway is now referred to as the Fanconi Anemia/BRCA pathway or network (D'Andrea).

#### **3.3 BRCA2 in cell cycle signaling and the DNA damage response**

To activate BRCA2, a sequence of cell signaling events is initiated in response to DNA damage, called the DNA damage response (or DDR). When the cell has experienced DSBs, either by exogenous sources such as ionizing radiation or exposure to crosslinking agents such as cisplatin, or endogenous sources such as free radicals and stalled replication forks, the goal is to immediately arrest cell division and repair the damage. When efficient DNA repair does not occur, apoptosis is induced to prevent propagation of genetic mutations. The phosphoinositide 3-kinase related kinases (PIKKs), ataxia-telangiectasia mutated kinase (ATM), and ATM and Rad 3-related kinase (ATR), are crucial in the detection and subsequent resolution of DNA damage. Furthermore, they are also involved in the Fanconi Anemia pathway, as previously described. ATM and ATR "cross-talk" with each other, given that ATM activates ATR in response to ionizing radiation, and ATR activates ATM in response to ultraviolet light. With respect to the DDR pathway that involves BRCA2, resolution of DSBs is initiated by activation of ATR, after phosphorylation by ATM. ATR proceeds to phosphorylate and active Chk1, which then phosphorylates RAD51. Chk1 arrests the cells in S and G2 phases to ensure DNA is repaired before synthesis and cell division. At this point, RAD51 is now able to engage in HR-mediated repair of DSBs under the regulation of BRCA2 (McNeely, Conti et al.), (Connell, Shibata et al.).

BRCA2 appears to play a crucial role during S and G2 phases of the cell cycle. First, during S phase, replication forks can stall and collapse due to exogenous or endogenous sources of damage. A DNA strand break at a replication fork can mimic a DSB as a result of the nascent DNA chain that is being synthesized at the fork. At this point, activated RAD51 is required to repair the break and subsequently stalled fork. It has been proposed that deficient BRCA2, which functions to regulate RAD51 during HR-mediated repair, may be a major cause of diseases resulting from an accumulation of stalled replication forks and consequential DNA breaks that remain unrepaired (Lomonosov, Anand et al. 2003). And, with respect to G2 phase, in a study where the binding of BRCA2 with RAD51 was inhibited in cells expressing the BRC4 repeat, which competed against endogenous full-length BRCA2, there was a failure to initiate radiation-induced G2/M checkpoint arrest. These results implied that the interaction between BRCA2 and RAD51 was imperative for G2/M checkpoint control (Chen, Chen et al. 1999).

The majority of *BRCA2* mutations that are associated with cancer predisposition tend be truncations that remove substantial portions of the CTD, which is where the domains required for DNA repair are located. This region of the protein also appears to be significant in cell cycle changes due to the DNA damage response, via the TR2 domain. The TR2 domain contains a serine at 3291 that is CDK phosphorylated and appears to be one method

*BRCA2* Mutations and Consequences for DNA Repair 335

A great deal of attention has been focused on the role of the C-terminus and BRC repeat region of BRCA2 in HR-mediated repair. However, mutations of the N-terminus also have detrimental effects on protein function. The N-terminus of BRCA2 binds the protein PALB2 (partner and localizer of BRCA2) (Xia, Sheng et al. 2006); (Rahman, Seal et al. 2007), (Figure 2). PALB2 is also a member of the Fanconi Anemia pathway, denoted as FANCN, in the same manner in which BRCA2/FANCD1 is involved, as well (Figure 3). And, just as biallelic mutations of BRCA2/FANCD1 cause a subtype of Fanconi Anemia, and susceptibility to childhood cancers, biallelic mutations of PALB2/FANCN have a similar phenotype (D'Andrea)**.** With respect to the interaction with BRCA2, PALB2 is responsible for localizing BRCA2 to the sites of DNA damage in order to promote repair (Xia, Sheng et al. 2006). Mutations of the PALB2 binding site on BRCA2 prevent this interaction, causing impaired formation of RAD51 damage-induced foci, and unresolved DSBs (Xia, Sheng et al. 2006). Furthermore, PALB2 is also able to bind DNA and enhance the recombination activity of

Cells that are defective in BRCA1 and BRCA2 retain unresolved DSBs. This attribute, which is detrimental in terms of genomic instability and risk for cancer, is actually a potent target for inhibitors of Poly(ADP-ribose) polymerase, or PARP, in the eradication of transformed cells.

PARPs are a family of 17 enzymes, with PARP-1 and -2 having been shown to be involved in DNA repair. PARP-1 is a nuclear protein with a zinc-finger DNA binding domain (Amir, Seruga et al.). It is responsible for binding to the sites of single-strand breaks, signaling damage at the site, and the initiating repair. The zinc finger domain binds to ssDNA breaks, cleaves NAD+, and attaches multiple ADP-ribose units to the protein. This results in an extremely negatively charged target which causes unwinding of the damaged DNA, followed by repair by the Base-Excision Repair (BER) pathway (Schreiber, Dantzer et al. 2006); (Ratnam and Low 2007)**.** However, PARP-1 has also been shown to serve as an antirecombinogenic factor at sites of damage where it has bound, thereby having implications on inhibiting HR-mediated repair (Amir, Seruga et al.), (Sandhu, Yap et al.). BRCA1 and -2 mutant cells are defective in repair of DSBs, and as a consequence, are sensitive to agents that induce DSBs. PARP-1 inhibitors have been shown to be effective in selectively targeting BRCA1 and -2 defective cells by converting SSBs, which have been induced by the use of chemotherapeutic agents, ionizing radiation, or occurring in normal cellular processes, such as stalled replication forks, to DSBs. The SSBs would have normally been identified and resolved by PARP-1 binding and the BER pathway; however, PARP-1 inhibitors prevent such resolution, and during DNA synthesis, the SSBs are converted to DSBs. The DSBs are normally resolved by HR-mediated repair involving BRCA1, and most important BRCA2, with the recombinase RAD51. However, this is deficient in BRCA-mutant cells and the addition of PARP inhibition enhances DNA-damage induced cell cycle arrest and apoptosis.

The first PARP-1 inhibitor created was 3-aminobenzamide (3-AB). It causes inhibition of PARP-1 by competing with NAD+ as a substrate. However, 3-AB showed poor specificity

**4. Therapeutic regimens designed to target BRCA2 defects** 

**4.1 Efficacy of PARP inhibitors in treating BRCA2-tumors** 

RAD51 (Dray, Etchin et al.).

This process eradicates the tumor cells.

**4.2 Development of PARP inhibitors** 

in which binding between BRCA2 and RAD51 is regulated (Esashi, Christ et al. 2005). There is reduced phosphorylation at this site during S phase, which allows BRCA2 and RAD51 to interact, and engage in HR-mediated repair resulting from replication-induced DNA breaks. In addition, phosphorylation is reduced in response to ionizing radiation. However, phosphorylation of S3291 increases during G2/M to inactivate HR from occurring during mitosis. Further support for this region of the protein being a cancer-related mutation site is evidenced by the association of the P3292L mutation with breast cancer incidence (Esashi, Christ et al. 2005). The TR2 domain also only interacts with multimeric forms of RAD51, both in the presence and absence of DNA (Esashi, Galkin et al. 2007). And, RAD51 monomers bearing mutations that prevent self-association do not interact with the TR2 domain. The impact that this has on BRCA2 function is quite remarkable and has been elegantly summarized (Esashi, Galkin et al. 2007). In the absence of DNA damage, the TR2 domain is phosphorylated at S3291, preventing association of the C-terminus of BRCA2 with RAD51, as well as keeping BRCA2 inactive. However, concurrently, RAD51 is associated with BRCA2 via the BRC repeats in monomeric form. And, it has been noted that the BRC repeats may serve as a negative regulator of RAD51 by preventing it from forming nucleoprotein filaments with ssDNA until after damage has been detected and the DNA has been prepped for HR-mediated repair. After DNA damage has been detected, S3291 is dephosphorylated, now allowing BRCA2 to become activated. The C-terminus can now bind with RAD51 in multimeric form, and the OB folds which possess ssDNA binding activity, deliver RAD51 to sites of DNA damage. This change in BRCA2 function from negatively regulating RAD51 to mobilizing it to sites of damage may be driven by the selfassembly of RAD51 from a monomeric to a multimeric state in response to DNA damage (Esashi, Galkin et al. 2007). This detrimental function of the C-terminus of BRCA2 further substantiates how truncations of this region of the protein, which are commonly seen in BRCA2-cancers, incite genomic instability and subsequent malignant transformation.

The role of the BRC repeat region has been somewhat controversial. It has been described as the region of BRCA2 that is responsible for delivering RAD51 to ssDNA at sites of DNA damage (Carreira, Hilario et al. 2009), (Shivji, Davies et al. 2006) but, conversely, as a negative regulator of RAD51, which was described in the previous section (Nomme, Takizawa et al. 2008), (Davies and Pellegrini 2007). Results of a study investigating cancer-associated mutations of BRC repeats supported their role as a negative regulator that binds and inhibits RAD51 from engaging in HR. But, then releases RAD51 monomers upon detection of DNA damage, thus allowing RAD51 to multimerize and interact with the BRCA2 TR2 region for mobilization to sites of damage. At this point, RAD51 is ready to form nucleoprotein filaments on the 3' ssDNA overhangs at the breakpoint junction, which will invade the DNA homologue to be used as the template for repair. Considering that the BRC repeats are important for modulating RAD51 activity, several cancerassociated mutations, primarily point mutations, have been identified in this highly conserved region of BRCA2. Cancer-associated mutations have been identified in BRC motifs 1(T1011R), 2(F1219L, S1221P), 4(G1529R), and 7(T1980I) (Esashi, Galkin et al. 2007). The effect of mutations in BRC motifs 2 and 4 on RAD51-mediated HR repair was assessed. The results determined that such mutations prevent binding of monomeric RAD51 to the BRC repeats, which prevents recruitment of RAD51 to DSBs, thereby inhibiting nucleoprotein filament formation and impairing HR-mediated repair (Tal, Arbel-Goren et al. 2009).

in which binding between BRCA2 and RAD51 is regulated (Esashi, Christ et al. 2005). There is reduced phosphorylation at this site during S phase, which allows BRCA2 and RAD51 to interact, and engage in HR-mediated repair resulting from replication-induced DNA breaks. In addition, phosphorylation is reduced in response to ionizing radiation. However, phosphorylation of S3291 increases during G2/M to inactivate HR from occurring during mitosis. Further support for this region of the protein being a cancer-related mutation site is evidenced by the association of the P3292L mutation with breast cancer incidence (Esashi, Christ et al. 2005). The TR2 domain also only interacts with multimeric forms of RAD51, both in the presence and absence of DNA (Esashi, Galkin et al. 2007). And, RAD51 monomers bearing mutations that prevent self-association do not interact with the TR2 domain. The impact that this has on BRCA2 function is quite remarkable and has been elegantly summarized (Esashi, Galkin et al. 2007). In the absence of DNA damage, the TR2 domain is phosphorylated at S3291, preventing association of the C-terminus of BRCA2 with RAD51, as well as keeping BRCA2 inactive. However, concurrently, RAD51 is associated with BRCA2 via the BRC repeats in monomeric form. And, it has been noted that the BRC repeats may serve as a negative regulator of RAD51 by preventing it from forming nucleoprotein filaments with ssDNA until after damage has been detected and the DNA has been prepped for HR-mediated repair. After DNA damage has been detected, S3291 is dephosphorylated, now allowing BRCA2 to become activated. The C-terminus can now bind with RAD51 in multimeric form, and the OB folds which possess ssDNA binding activity, deliver RAD51 to sites of DNA damage. This change in BRCA2 function from negatively regulating RAD51 to mobilizing it to sites of damage may be driven by the selfassembly of RAD51 from a monomeric to a multimeric state in response to DNA damage (Esashi, Galkin et al. 2007). This detrimental function of the C-terminus of BRCA2 further substantiates how truncations of this region of the protein, which are commonly seen in

BRCA2-cancers, incite genomic instability and subsequent malignant transformation.

Arbel-Goren et al. 2009).

The role of the BRC repeat region has been somewhat controversial. It has been described as the region of BRCA2 that is responsible for delivering RAD51 to ssDNA at sites of DNA damage (Carreira, Hilario et al. 2009), (Shivji, Davies et al. 2006) but, conversely, as a negative regulator of RAD51, which was described in the previous section (Nomme, Takizawa et al. 2008), (Davies and Pellegrini 2007). Results of a study investigating cancer-associated mutations of BRC repeats supported their role as a negative regulator that binds and inhibits RAD51 from engaging in HR. But, then releases RAD51 monomers upon detection of DNA damage, thus allowing RAD51 to multimerize and interact with the BRCA2 TR2 region for mobilization to sites of damage. At this point, RAD51 is ready to form nucleoprotein filaments on the 3' ssDNA overhangs at the breakpoint junction, which will invade the DNA homologue to be used as the template for repair. Considering that the BRC repeats are important for modulating RAD51 activity, several cancerassociated mutations, primarily point mutations, have been identified in this highly conserved region of BRCA2. Cancer-associated mutations have been identified in BRC motifs 1(T1011R), 2(F1219L, S1221P), 4(G1529R), and 7(T1980I) (Esashi, Galkin et al. 2007). The effect of mutations in BRC motifs 2 and 4 on RAD51-mediated HR repair was assessed. The results determined that such mutations prevent binding of monomeric RAD51 to the BRC repeats, which prevents recruitment of RAD51 to DSBs, thereby inhibiting nucleoprotein filament formation and impairing HR-mediated repair (Tal, A great deal of attention has been focused on the role of the C-terminus and BRC repeat region of BRCA2 in HR-mediated repair. However, mutations of the N-terminus also have detrimental effects on protein function. The N-terminus of BRCA2 binds the protein PALB2 (partner and localizer of BRCA2) (Xia, Sheng et al. 2006); (Rahman, Seal et al. 2007), (Figure 2). PALB2 is also a member of the Fanconi Anemia pathway, denoted as FANCN, in the same manner in which BRCA2/FANCD1 is involved, as well (Figure 3). And, just as biallelic mutations of BRCA2/FANCD1 cause a subtype of Fanconi Anemia, and susceptibility to childhood cancers, biallelic mutations of PALB2/FANCN have a similar phenotype (D'Andrea)**.** With respect to the interaction with BRCA2, PALB2 is responsible for localizing BRCA2 to the sites of DNA damage in order to promote repair (Xia, Sheng et al. 2006). Mutations of the PALB2 binding site on BRCA2 prevent this interaction, causing impaired formation of RAD51 damage-induced foci, and unresolved DSBs (Xia, Sheng et al. 2006). Furthermore, PALB2 is also able to bind DNA and enhance the recombination activity of RAD51 (Dray, Etchin et al.).
