**3. Rad51-interacting proteins involved in the nuclear translocation of Rad51 and in the HR process**

The number and size of Rad51 nuclear foci is a hallmark of the cellular response to genotoxic stress. These nuclear foci characterize the formation of Rad51 filaments. Indeed Rad51 is recruited to sites of DNA DSBs in response to damage where it promotes DNA strand invasion and strand exchange. Impaired formation of Rad51 foci in response to DNA damage has been demonstrated in hamster or chicken cells defective in the Rad51 paralogs XRCC2, XRCC3, Rad51B, Rad51C, and in mammalian BRCA1 or BRCA2-defective cells (Chen et al., 1999c; Takata et al., 2001; Yuan et al., 1999).

The foci formation requires the translocation of Rad51 into the nucleus after DSB induction by genotoxic stress or stalled replication forks (Haaf et al., 1995).) This process is often accompanied by posttranslational modifications of Rad51 partners which cooperate to achieve the fidelity of DNA repair. Several works have shown that these modifications can modulate protein interactions involving Rad51 and can affect Rad51 foci formation.

#### **3.1 Nuclear translocation of Rad51**

The first stage of DNA DSB repair by HR requires the delivery of Rad51 at the sites of DNA damage. Since Rad51 does not have a Nuclear Localisation Signal (NLS) sequence, its nuclear entry likely requires the interaction with other proteins containing functional NLS sequences (Gildemeister et al., 2009). BRCA1 and BRCA2 proteins have both been described as primordial recombination mediators for the nuclear translocation of Rad51.

#### **3.1.1 Involvement of BRCA1/Akt1**

Several studies have demonstrated that the overexpression of Rad51 results in its cytoplasmic accumulation (Mladenov et al., 2006) but genotoxic stress triggers the translocation of Rad51 from the cytoplasm to the nucleus (Gildemeister et al., 2009). Plo and

Posttranslational Modifications of Rad51 Protein and Its Direct Partners:

interaction with another co-factor, Rad51AP1 (Dray et al., 2010).

of RPA in the presynaptic assembly of Rad51 (Sleeth et al., 2007).

nucleofilament formation (McIlwraith et al., 2000).

Wong et al., 1997).

elsewhere (Dong et al., 2003).

with Rad51, PALB2 and DNA.

sites of DSB and in initiating the HR process.

Role and Effect on Homologous Recombination – Mediated DNA Repair 151

This results in the generation of 3' single-stranded DNA (ssDNA). RPA (Replication Protein A) binds to the 3' overhangs and thus protects them from further resection. This protein also removes secondary structures present on the ssDNA which allows efficient Rad51

During the presynaptic phase Rad51 is loaded on the ssDNA ends with the help of BRCA2 (Huen et al., 2010). Rad51 recognizes and binds to the BRC repeats and the TR2 domain of BRCA2 (Fig.2). The Oligonucleotide Binding Folds (OB Folds) in the C-terminal region of the protein are also required for the recruitment of Rad51 (O'Donovan and Livingston, 2010;

The interaction of BRCA2 with two other proteins, BRCA1 and the bridging factor PALB2, is necessary for its role in the presynaptic phase of HR. These proteins along with other factors form a macro-complex named BRCC whose role in DNA repair has been described

In addition to its linking function between BRCA1 and BRCA2, PALB2 also interacts with a domain in Rad51 which is comprised between amino acids 184 and 257 (Fig.3) (Buisson et al., 2010). Thus, PALB2 cooperates with BRCA2 to stimulate Rad51 filament assembly during HR. The stimulation of the filament assembly by PALB2 is also mediated by its

Fig. 2. Domain organization of BRCA2. Schematic drawing indicating the interaction sites

According to these data, BRCA2 plays an essential role in recruiting and loading Rad51 on

In order for the Rad51 presynaptic filament to assemble, Rad52 has to displace RPA from the ssDNA (Sugiyama and Kowalczykowski, 2002). RPA is a single-stranded DNA binding protein composed of three subunits, with sizes of respectively 70, 32 and 14 kDa (Wold, 1997). It has previously been shown by co-immunoprecipitation experiments that each of the three subunits of RPA interacts with Rad51, and that the RPA-Rad51 interaction is regulated by the 70kDa subunit (Golub et al., 1998). The co-localization of Rad51 and RPA foci in response to ionizing radiation was observed in a mice fibroblast model and suggests a possible *in vivo* interaction between the two proteins. Furthermore, a recent study has shown that depletion of RPA in mammalian cells leads to the impairment of Rad51 foci formation following DSB induced by hydroxyurea treatment. This confirms the importance

Because RPA binding on ssDNA may prevent Rad51 access to DSB, the presynaptic filament formation needs to be time-regulated by the mediator Rad52. Rad52 is a key member of the RAD52 epistasis group, which includes Rad51, and whose function in HR has been previously described (Symington, 2002). The human Rad52 (hRad52) protein contains 418 amino acids. It has a highly conserved region in its N-terminus, and possesses a

colleagues have reported that the nuclear translocation of Rad51 was impaired by AKT1 which repressed HR (Plo et al., 2008). In tumour cells with high levels of active AKT1, BRCA1 and Rad51 are retained in the cytoplasm. However, BRCA1 phosphorylation by AKT1 was not required for this retention. Interestingly, 77% of tumours containing high levels of AKT1 exhibited also cytoplasmic retention of Rad51 (Plo et al., 2008). This shows that AKT1 activation strongly favors the cytoplasmic localization of both BRCA1 and Rad51 proteins.

#### **3.1.2 BRCA2-mediated nuclear translocation of Rad51**

Like BRCA1, BRCA2 is a tumour suppressor implicated in familial breast cancer. BRCA2 protein contains six highly conserved BRC repeats which are involved in the interaction between BRCA2 and Rad51 (Marmorstein et al., 1998; Mizuta et al., 1997; Wong et al., 1997). It has been proposed that the BRCA2 protein is directly involved in the regulation of the nucleofilament formation and in the nuclear transport of Rad51 (Davies et al., 2001).

Medova and colleagues have demonstrated that the inhibition of the MET receptor tyrosine kinase by a small inhibitor molecule impairs the formation of the Rad51-BRCA2 complex. By targeting MET, the authors have shown the incapacity of tumour cells to repair DNA DSBs through homologous recombination. This was due to the impaired translocation of Rad51 into the nucleus (Medova et al.).

The pancreatic adenocarcinoma cell line CAPAN-1 is the best characterized BRCA2 defective human cell line (Jasin, 2002). CAPAN-1 cells have indeed lost a wild-type BRCA2 allele and presents a 6174delT mutation on the other allele. This mutation causes the premature C-terminal truncation of the protein. This results in the deletion of the BRCA2 domains for DNA repair and the nuclear localization signals (Holt et al., 2008). Rad51 exhibits impaired nuclear translocation in CAPAN-1 cells. Therefore it has been proposed that Rad51 requires BRCA2 for its nuclear translocation and that C-terminally truncated BRCA2 retains Rad51 in the cytoplasm.

Another group has however observed a DNA damage-induced increase in nuclear Rad51 in the BRCA2-defective cell line CAPAN-1. Moreover, chromatin-associated Rad51 levels were found to be increased (2-fold) following IR exposure (Gildemeister et al., 2009).

To analyze a possible BRCA2-independent mechanism for Rad51 nuclear transport, the authors studied two other Rad51-interacting proteins, Rad51C and Xrcc3. Both of these proteins contain a functional NLS. In contrast to Xrcc3, subcellular distribution of Rad51C was affected by DNA damage since nuclear Rad51C was significantly increased following IR exposure. Furthermore, the depletion of Rad51C in HeLa and CAPAN-1 cells by RNA interference resulted in lower levels of nuclear Rad51. These results provide an important overview of the cellular regulation of Rad51 nuclear entry. This data underlines the potential role for Rad51C in the nuclear translocation of Rad51, which suggests a BRCA2 independent mechanism for Rad51 nuclear entry both before and after DNA damage. Other studies have also demonstrated that an interaction between Rad51 and BRCA2 is not required for nuclear transport of Rad51 but it may prevent the formation of Rad51 filaments in the cytoplasm.

#### **3.2 Recruitment of Rad51 at the damage site – Presynaptic phase of HR**

Following damage, DSB are recognized by the MRN complex (MRE11-Rad51-NSB1 complex). MRN binds to and resects the extremities of the DSB through its nuclease activity.

colleagues have reported that the nuclear translocation of Rad51 was impaired by AKT1 which repressed HR (Plo et al., 2008). In tumour cells with high levels of active AKT1, BRCA1 and Rad51 are retained in the cytoplasm. However, BRCA1 phosphorylation by AKT1 was not required for this retention. Interestingly, 77% of tumours containing high levels of AKT1 exhibited also cytoplasmic retention of Rad51 (Plo et al., 2008). This shows that AKT1 activation strongly favors the cytoplasmic localization of both BRCA1 and Rad51

Like BRCA1, BRCA2 is a tumour suppressor implicated in familial breast cancer. BRCA2 protein contains six highly conserved BRC repeats which are involved in the interaction between BRCA2 and Rad51 (Marmorstein et al., 1998; Mizuta et al., 1997; Wong et al., 1997). It has been proposed that the BRCA2 protein is directly involved in the regulation of the

Medova and colleagues have demonstrated that the inhibition of the MET receptor tyrosine kinase by a small inhibitor molecule impairs the formation of the Rad51-BRCA2 complex. By targeting MET, the authors have shown the incapacity of tumour cells to repair DNA DSBs through homologous recombination. This was due to the impaired translocation of

The pancreatic adenocarcinoma cell line CAPAN-1 is the best characterized BRCA2 defective human cell line (Jasin, 2002). CAPAN-1 cells have indeed lost a wild-type BRCA2 allele and presents a 6174delT mutation on the other allele. This mutation causes the premature C-terminal truncation of the protein. This results in the deletion of the BRCA2 domains for DNA repair and the nuclear localization signals (Holt et al., 2008). Rad51 exhibits impaired nuclear translocation in CAPAN-1 cells. Therefore it has been proposed that Rad51 requires BRCA2 for its nuclear translocation and that C-terminally truncated

Another group has however observed a DNA damage-induced increase in nuclear Rad51 in the BRCA2-defective cell line CAPAN-1. Moreover, chromatin-associated Rad51 levels were

To analyze a possible BRCA2-independent mechanism for Rad51 nuclear transport, the authors studied two other Rad51-interacting proteins, Rad51C and Xrcc3. Both of these proteins contain a functional NLS. In contrast to Xrcc3, subcellular distribution of Rad51C was affected by DNA damage since nuclear Rad51C was significantly increased following IR exposure. Furthermore, the depletion of Rad51C in HeLa and CAPAN-1 cells by RNA interference resulted in lower levels of nuclear Rad51. These results provide an important overview of the cellular regulation of Rad51 nuclear entry. This data underlines the potential role for Rad51C in the nuclear translocation of Rad51, which suggests a BRCA2 independent mechanism for Rad51 nuclear entry both before and after DNA damage. Other studies have also demonstrated that an interaction between Rad51 and BRCA2 is not required for nuclear transport of Rad51 but it may prevent the formation of Rad51 filaments

Following damage, DSB are recognized by the MRN complex (MRE11-Rad51-NSB1 complex). MRN binds to and resects the extremities of the DSB through its nuclease activity.

found to be increased (2-fold) following IR exposure (Gildemeister et al., 2009).

**3.2 Recruitment of Rad51 at the damage site – Presynaptic phase of HR** 

nucleofilament formation and in the nuclear transport of Rad51 (Davies et al., 2001).

**3.1.2 BRCA2-mediated nuclear translocation of Rad51** 

Rad51 into the nucleus (Medova et al.).

BRCA2 retains Rad51 in the cytoplasm.

in the cytoplasm.

proteins.

This results in the generation of 3' single-stranded DNA (ssDNA). RPA (Replication Protein A) binds to the 3' overhangs and thus protects them from further resection. This protein also removes secondary structures present on the ssDNA which allows efficient Rad51 nucleofilament formation (McIlwraith et al., 2000).

During the presynaptic phase Rad51 is loaded on the ssDNA ends with the help of BRCA2 (Huen et al., 2010). Rad51 recognizes and binds to the BRC repeats and the TR2 domain of BRCA2 (Fig.2). The Oligonucleotide Binding Folds (OB Folds) in the C-terminal region of the protein are also required for the recruitment of Rad51 (O'Donovan and Livingston, 2010; Wong et al., 1997).

The interaction of BRCA2 with two other proteins, BRCA1 and the bridging factor PALB2, is necessary for its role in the presynaptic phase of HR. These proteins along with other factors form a macro-complex named BRCC whose role in DNA repair has been described elsewhere (Dong et al., 2003).

In addition to its linking function between BRCA1 and BRCA2, PALB2 also interacts with a domain in Rad51 which is comprised between amino acids 184 and 257 (Fig.3) (Buisson et al., 2010). Thus, PALB2 cooperates with BRCA2 to stimulate Rad51 filament assembly during HR. The stimulation of the filament assembly by PALB2 is also mediated by its interaction with another co-factor, Rad51AP1 (Dray et al., 2010).

Fig. 2. Domain organization of BRCA2. Schematic drawing indicating the interaction sites with Rad51, PALB2 and DNA.

According to these data, BRCA2 plays an essential role in recruiting and loading Rad51 on sites of DSB and in initiating the HR process.

In order for the Rad51 presynaptic filament to assemble, Rad52 has to displace RPA from the ssDNA (Sugiyama and Kowalczykowski, 2002). RPA is a single-stranded DNA binding protein composed of three subunits, with sizes of respectively 70, 32 and 14 kDa (Wold, 1997). It has previously been shown by co-immunoprecipitation experiments that each of the three subunits of RPA interacts with Rad51, and that the RPA-Rad51 interaction is regulated by the 70kDa subunit (Golub et al., 1998). The co-localization of Rad51 and RPA foci in response to ionizing radiation was observed in a mice fibroblast model and suggests a possible *in vivo* interaction between the two proteins. Furthermore, a recent study has shown that depletion of RPA in mammalian cells leads to the impairment of Rad51 foci formation following DSB induced by hydroxyurea treatment. This confirms the importance of RPA in the presynaptic assembly of Rad51 (Sleeth et al., 2007).

Because RPA binding on ssDNA may prevent Rad51 access to DSB, the presynaptic filament formation needs to be time-regulated by the mediator Rad52. Rad52 is a key member of the RAD52 epistasis group, which includes Rad51, and whose function in HR has been previously described (Symington, 2002). The human Rad52 (hRad52) protein contains 418 amino acids. It has a highly conserved region in its N-terminus, and possesses a

Posttranslational Modifications of Rad51 Protein and Its Direct Partners:

**exchange activity - Synaptic phase** 

stability of the Rad51 filament.

al., 2007).

eukaryotes.

Role and Effect on Homologous Recombination – Mediated DNA Repair 153

Once the Rad51 nucleofilament is assembled, it has to be stabilized before Rad51 strand exchange activity may occur. This is mainly achieved by the Rad54 protein, which interacts both *in vitro* and *in vivo* with Rad51 during the synaptic phase of HR (Golub et al., 1997; Mazin et al., 2010). This protein-protein interaction is mediated by the Rad54 N-terminal region. It can occur either with the free Rad51 protein or with the assembled nucelofilament (Mazin et al., 2003; Raschle et al., 2004). Furthermore, using mouse embryonic stem cells, Tan and colleagues have demonstrated that Rad54 is required for Rad51 IR-induced foci formation (Tan et al., 1999). Rad54 functions in an ATP-independent manner to stabilize the Rad51 nucleofilament (Wolner and Peterson, 2005). However, it can also disrupt the assembled Rad51 complex (Li et al., 2007; Solinger et al., 2002). Thus, Rad54 modulates the

Another important consequence of the Rad51-Rad54 interaction is that Rad54 stimulates the recombinase and strand exchange activities of Rad51 (Mazina and Mazin, 2004; Sigurdsson et al., 2002). An additional protein interacting with Rad51 in the mature synaptic filament has been discovered. First identified as Pir51 (for Protein interacting with Rad51), this cofactor was later renamed Rad51AP1 (Rad51 Associated Protein 1). This protein was first characterized for its DNA crosslink repair activity (Henson et al., 2006; Kovalenko et al., 1997). Modesti and colleagues proposed a model in which Rad51AP1 could stimulate the formation of the D-loop by Rad51, which is the final step of the synaptic phase (Modesti et

To this day, the potential effect of Rad54 posttranslational modifications on Rad51 activity during this late stage of HR has not been demonstrated. Recent results obtained in yeast show that Rad54 phosphorylation leads to a reduction in Rad51-Rad54 complexes (Niu et al., 2009). It is not excluded that a similar mechanism could exist in superior

Following the synaptic phase, D-loops can be eliminated by different subpathways, each requiring different proteins. Here we will present only the pathways involving double Holliday junctions (dHJ) (Bzymek et al., 2010). Double HJ are structural intermediates which are resolved by specific endonucleases and result in either crossover or non-crossover products. The dHJ intermediates can also be resolved by helicases (RecQ helicase family) combined with topoisomerase action. In human cells, this pathway combines BLM helicase and topoisomerase IIIa, both of which catalyze dHJ dissolution (Wu and Hickson, 2003). Interestingly, BLM helicase is phosphorylated by different kinases, such as Chk1, at different stages of the cell cycle or in response to DNA damage. BLM can interact with 53PB1, a signal transducer, and with Topoisomerase IIIa during the presynaptic and the postsynaptic phases of HR respectively. It has been shown that BLM and 53BP1 can interact physically with Rad51 and regulate HR by modulating the assembly of Rad51 filaments. The *in vivo* phosphorylation of both BLM and 53BP1 affects negatively Rad51 foci formation (Tripathi et al., 2007). Concerning Topoisomerase IIIa, Rao and colleagues suggested that the BLM phosphorylation on T99 results in its dissociation from topoisomerase IIIa, thereby

**3.4 Post-synaptic phase of HR – Resolution of Holliday junction** 

modulating the resolution of dHJ (Rao et al., 2005).

**3.3 Regulation of Rad51 nucleofilament stability and enhancement of the strand** 

ssDNA/dsDNA binding region and a RPA binding site (Kagawa et al., 2002; Park et al., 1996). Shen and colleagues have demonstrated both *in vitro* and *in vivo* that hRad52 physically interacts with hRad51. The Rad51 binding domain on Rad52 has been identified between residues 291 to 330 (Fig.3) located in the C-terminal region of the protein (Shen et al., 1996).

Furthermore, five amino acid residues of hRad51 have been shown to participate in the Rad51-Rad52 interaction. These residues are located in the C-terminal region of hRad51 (Kurumizaka et al., 1999). Interestingly, the Rad52 binding site on Rad51 is not the same in *Homo Sapiens* and *Saccahromyces cerevisiae,* suggesting that this interaction is not conserved among species.

Fig. 3. Human Rad52 (hRad52) domains involved in HR.

The capacity to bind RPA and DNA confers to Rad52 the ability to displace RPA from the ssDNA and thus helps the formation of the Rad51 presynaptic filament (Plate et al., 2008; San Filippo et al., 2008).

The posttranslational modifications of RPA and Rad52 could modulate the formation of the presynaptic filament. Indeed, RPA is phosphorylated on one of its three subunits in a DNA damage-dependent manner and the resulting hyperphosphorylated RPA proteins directly interact with Rad51 (Binz et al., 2004; Wu et al., 2005). More recently, Shi and colleagues demonstrated by mutating the phosphorylation site of RPA that this posttranslational modification is required for Rad51 assembly (Shi et al., 2010). The importance of RPA phosphorylation during the presynaptic phase of HR was confirmed by Deng and colleagues who proposed a model in which RPA phosphorylation promotes Rad52 function and thus prepares DSB to be processed by Rad51 (Deng et al., 2009).

Phosphorylation of the Rad52 mediator in a c-Abl dependant manner has also been described in response to ionizing treatment (Kitao and Yuan, 2002). There is no evidence for the direct effect of Rad52 phosphorylation on Rad51 assembly. However, anterior studies have shown that the phosphorylation of Rad51 by c-Abl has an impact on the interaction between Rad51 and Rad52 (Chen et al., 1999b).

Another important posttranslational modification which plays a role in this stage of the HR process is SUMOylation. SUMOylation is already known to regulate the properties and stability of different proteins (Hay, 2005). It has recently been shown that the 70 kDa subunit of RPA can be SUMOylated and this process may regulate Rad51 presynaptic filament formation (Dou et al., 2010).

ssDNA/dsDNA binding region and a RPA binding site (Kagawa et al., 2002; Park et al., 1996). Shen and colleagues have demonstrated both *in vitro* and *in vivo* that hRad52 physically interacts with hRad51. The Rad51 binding domain on Rad52 has been identified between residues 291 to 330 (Fig.3) located in the C-terminal region of the protein (Shen et

Furthermore, five amino acid residues of hRad51 have been shown to participate in the Rad51-Rad52 interaction. These residues are located in the C-terminal region of hRad51 (Kurumizaka et al., 1999). Interestingly, the Rad52 binding site on Rad51 is not the same in *Homo Sapiens* and *Saccahromyces cerevisiae,* suggesting that this interaction is not conserved

The capacity to bind RPA and DNA confers to Rad52 the ability to displace RPA from the ssDNA and thus helps the formation of the Rad51 presynaptic filament (Plate et al., 2008;

The posttranslational modifications of RPA and Rad52 could modulate the formation of the presynaptic filament. Indeed, RPA is phosphorylated on one of its three subunits in a DNA damage-dependent manner and the resulting hyperphosphorylated RPA proteins directly interact with Rad51 (Binz et al., 2004; Wu et al., 2005). More recently, Shi and colleagues demonstrated by mutating the phosphorylation site of RPA that this posttranslational modification is required for Rad51 assembly (Shi et al., 2010). The importance of RPA phosphorylation during the presynaptic phase of HR was confirmed by Deng and colleagues who proposed a model in which RPA phosphorylation promotes Rad52 function

Phosphorylation of the Rad52 mediator in a c-Abl dependant manner has also been described in response to ionizing treatment (Kitao and Yuan, 2002). There is no evidence for the direct effect of Rad52 phosphorylation on Rad51 assembly. However, anterior studies have shown that the phosphorylation of Rad51 by c-Abl has an impact on the interaction

Another important posttranslational modification which plays a role in this stage of the HR process is SUMOylation. SUMOylation is already known to regulate the properties and stability of different proteins (Hay, 2005). It has recently been shown that the 70 kDa subunit of RPA can be SUMOylated and this process may regulate Rad51 presynaptic filament

Fig. 3. Human Rad52 (hRad52) domains involved in HR.

and thus prepares DSB to be processed by Rad51 (Deng et al., 2009).

between Rad51 and Rad52 (Chen et al., 1999b).

formation (Dou et al., 2010).

al., 1996).

among species.

San Filippo et al., 2008).

#### **3.3 Regulation of Rad51 nucleofilament stability and enhancement of the strand exchange activity - Synaptic phase**

Once the Rad51 nucleofilament is assembled, it has to be stabilized before Rad51 strand exchange activity may occur. This is mainly achieved by the Rad54 protein, which interacts both *in vitro* and *in vivo* with Rad51 during the synaptic phase of HR (Golub et al., 1997; Mazin et al., 2010). This protein-protein interaction is mediated by the Rad54 N-terminal region. It can occur either with the free Rad51 protein or with the assembled nucelofilament (Mazin et al., 2003; Raschle et al., 2004). Furthermore, using mouse embryonic stem cells, Tan and colleagues have demonstrated that Rad54 is required for Rad51 IR-induced foci formation (Tan et al., 1999). Rad54 functions in an ATP-independent manner to stabilize the Rad51 nucleofilament (Wolner and Peterson, 2005). However, it can also disrupt the assembled Rad51 complex (Li et al., 2007; Solinger et al., 2002). Thus, Rad54 modulates the stability of the Rad51 filament.

Another important consequence of the Rad51-Rad54 interaction is that Rad54 stimulates the recombinase and strand exchange activities of Rad51 (Mazina and Mazin, 2004; Sigurdsson et al., 2002). An additional protein interacting with Rad51 in the mature synaptic filament has been discovered. First identified as Pir51 (for Protein interacting with Rad51), this cofactor was later renamed Rad51AP1 (Rad51 Associated Protein 1). This protein was first characterized for its DNA crosslink repair activity (Henson et al., 2006; Kovalenko et al., 1997). Modesti and colleagues proposed a model in which Rad51AP1 could stimulate the formation of the D-loop by Rad51, which is the final step of the synaptic phase (Modesti et al., 2007).

To this day, the potential effect of Rad54 posttranslational modifications on Rad51 activity during this late stage of HR has not been demonstrated. Recent results obtained in yeast show that Rad54 phosphorylation leads to a reduction in Rad51-Rad54 complexes (Niu et al., 2009). It is not excluded that a similar mechanism could exist in superior eukaryotes.
