**2.1.6 Model of sequential phosphorylation**

146 DNA Repair

BCR/Abl and Rad51 resulted in the constitutive phosphorylation of Rad51 on tyrosine. Rad51 was also phosphorylated by c-Abl after treatment of cells with cisplatin and mitomycin C. In order to determine the position of phosphorylation, the authors transiently co-expressed BCR/Abl and wild type or mutated Rad51 in cells. Tyrosine to phenylalanine mutations were performed at Tyrosine 54 or Tyrosine 315. The analysis of the Rad51 immunoprecipitates with an anti-phosphoTyrosine antibody revealed the phosphorylation of the wild type and the Y54F Rad51 protein. A substantial reduction in the phosphorylation level of Rad51 was observed when Y315 was mutated to phenylalanine, indicating that the majority of the phosphorylation of Rad51 occurred on Y315. To further confirm the phosphorylation of the Y315 residue, Slupianek and colleagues prepared an antiserum using a phosphorylated Y315 peptide. Western blots were then performed with lysates from cells overexpressiong Rad51 alone or with BCR/Abl. The antiserum did not recognize Rad51 when the protein was overexpressed in cells alone. In contrast, in cells co-expressing BCR/Abl a strong signal was observed. This confirms that the fusion tyrosine kinase BCR/Abl phosphorylates Rad51 on Tyrosine 315

The only other member of the c-Abl family, the kinase Arg, also phosphorylates Rad51. Arg shares considerable structural and sequence homology with c-Abl in the N-terminal SH3 and SH2 domains, as well as in the tyrosine kinase domain (Kruh et al., 1990). Coimmunoprecipitation of Rad51 from cells overexpressing Rad51 and Arg indicated that Arg can interact with Rad51 *in vivo*. An anti-phosphoTyrosine antibody showed that Rad51 is phosphorylated by Arg and this phosphorylation seemed to be more effective than the phosphorylation by c-Abl. However, the position of phosphorylation was not determined

The study conducted by Popova and colleagues has allowed to reconcile the discrepancies on which tyrosine residue is phosphorylated in Rad51. The authors purified specific antiphosphoTyrosine antibodies for each site of phosphorylation. These antibodies were used to analyze the phosphorylation state of Rad51 by immunoblotting of lysates from cells overexpressing Rad51 and c-Abl. The ability of these specific antibodies to detect distinctively the phosphorylation of the two tyrosine residues has allowed to observe the phosphorylation of both Y54 and Y315 in the same experiment. This confirmed that both

In all previous studies the phosphorylation of only one site was observed, either Y54 or Y315. The fact that Yuan and colleagues observed only the phosphorylation of Y54 and did not detect the phosphorylation of Y315 could be due to the technique they used. In their study, the *in vitro* or *in vivo* phosphorylated Rad51 protein, as well as the unphosphorylated protein were digested by trypsin. The obtained fragments were then analyzed by mass spectroscopy and the spectra of the unphosphorylated and the phosphorylated proteins were compared. The lack of a phosphorylation peak in the fragment containing Y315 could be explained by its biophysical characteristics. Following trypsin digestion, the peptide containing Tyrosine 54 is 17 amino acids long and has a pHi of 4,83. On the contrary, the peptide containing Tyrosine 315 is 28 amino acids long and its pHi is 4,03. Thus, the Y315 peptide is longer and more negatively charged compared to the Y54 peptide which could

**2.1.5 Phosphorylation of both Tyrosine 54 and Tyrosine 315 by c-Abl** 

Tyrosine 54 and 315 can be phosphorylated (Popova et al., 2009).

interfere with its detection by mass spectroscopy (Raggiaschi et al., 2005).

(Slupianek et al., 2001).

(Li et al., 2002).

**2.1.4 Phosphorylation by Arg** 

Popova and co-authors have established a possible mechanism by which Rad51 is phosphorylated by c-Abl. They co-expressed c-Abl and wild type or mutated hRad51 in cells. In the amino acid sequence of hRad51, Tyrosine 54 or Tyrosine 315 were mutated to phenylalanine, thus rendering the residue at this position nonphosphorylatable. Western blot analysis of the cell lysates, revealed with their specific anti-phosphoTyrosine antibodies, showed a relationship between the phosphorylation of Y54 and Y315. When residue 315 was mutated to phenylalanine and nonphosphorylatable, Tyrosine 54 was no longer phosphorylated. On the contrary, the mutation of residue 54 had no effect on the phosphorylation of Tyrosine 315. The authors hypothesized that the phosphorylation of Tyrosine 315 is needed for the phosphorylation of Tyrosine 54.

The c-Abl kinase possesses a SH3 and a SH2 domain in its N-terminal region. The SH3 domain recognizes and binds preferentially to proline rich regions containing the sequence PXXP. The SH2 domain recognizes pYXXP sequences. hRad51 has two PXXP motifs in its amino acid sequence – between amino acids 283 and 286, and between amino acids 318 and 321. When Tyrosine 315 is phosphorylated, a pYXXP motif is revealed between amino acids 315 and 318. This motif might be recognized by the SH2 domain of c-Abl.

According to this model of sequential phosphorylation, c-Abl recognizes a PXXP motif in the sequence of Rad51 through its SH3 domain and phosphorylates Tyrosine 315. The phosphorylation of this residue reveals the pYXXP binding motif which is recognized by the SH2 domain of c-Abl. This allows the phosphorylation of Tyrosine 54.

To confirm this model, GST pull-down assays were performed. A GST- c-Abl SH2 domain peptide was incubated with lysates from cells overexpressing Rad51 and c-Abl. The results showed that hRad51 binds to the SH2 domain of c-Abl and that this interaction takes place when Rad51 is phosphorylated on Tyrosine 315. Therefore a model of sequential phosphorylation of Rad51, where the phosphorylation of Tyrosine 315 by c-Abl reveals a novel binding site for the kinase thus allowing the phosphorylation of Tyrosine 54, is highly plausible.

#### **2.2 Role of Rad51 phosphorylation**

Even though the process of phosphorylation seems to be of considerable importance in the regulation of Rad51 activity, its exact roles and consequences have not been elucidated yet. Moreover, the existing data is contradictory.

In their study, Yuan and colleagues investigated the possible effect of Y54 phosphorylation on Rad51 activity. Strand exchange assays showed that phosphorylation of S. cerevisiae

Posttranslational Modifications of Rad51 Protein and Its Direct Partners:

**2.4 Sumoylation – Ubiquitination of Rad51** 

damage (Saitoh et al., 2002).

and resistance to IR (Li et al., 2000).

**Rad51 and in the HR process** 

**3.1 Nuclear translocation of Rad51** 

**3.1.1 Involvement of BRCA1/Akt1** 

(Chen et al., 1999c; Takata et al., 2001; Yuan et al., 1999).

Role and Effect on Homologous Recombination – Mediated DNA Repair 149

Yeast two-hybrid assays have shown that Rad51 can interact with HsUbc9, later named UBE21. HsUbc9/UBE21 is the human homologue of *S. cerevisiae* UBC9 and *S. pombe* Hus5 ubiquitin conjugating enzymes (Kovalenko et al., 1996; Shen et al., 1996). In mammalian cells the downregulation of Ubc9 was associated with defects in cytokinesis and an increased number of apoptotic cells. Furthermore, its gene inactivation is lethal in mouse embryos (Moschos and Mo, 2006). Nuclear depletion of Ubc9 disrupts the intracellular trafficking of Rad51 and thus inhibits the formation of Rad51 nuclear foci following DNA

Rad51 also interacts with UBL1 (ubiquitin like 1), also called PIC1, GMP1, SUMO-1 and Sentrin (Shen et al., 1996). The yeast homologue of UBL1, SMT3, inhibits a centrosome protein involved in centrosome segregation (Shen et al., 1996). UBL1 interacts with HsUBC9/UBE21 (Shen et al., 1996). Studies have shown that HsUbc9/UBE21 is a UBL1 conjugating enzyme, rather than an ubiquitin-conjugating enzyme. Immunoprecipitation essays in HeLa cells and GST pull-down essays have shown that the interaction between Rad51 and Ubl1 is mediated by Rad52 and/or Ubc9. This suggests that Ubc9 can conjugate UBL1 to Rad51. The overexpression of UBL1 in mammalian cells decreases DSB-induced HR

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

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

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

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

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

modulate protein interactions involving Rad51 and can affect Rad51 foci formation.

as primordial recombination mediators for the nuclear translocation of Rad51.

Rad51 (ScRad51) results in the inhibition of dsDNA conversion to joint molecules and nicked circular dsDNA. An inhibition of the binding of phospho-ScRad51 and phosphohRad51 to ssDNA was also observed. Because Rad51 exerts its activity by binding to and forming nucleofilaments with ssDNA, the authors concluded that by inhibiting the binding to ssDNA, phosphorylation inhibits Rad51 function (Yuan et al., 1998).

In the search of a possible role for Y315 phosphorylation, Chen and colleagues investigated if the phosphorylation impacts the interaction between Rad51 and Rad52. Rad52 is a protein needed in the presynaptic stage of homologous recombination (Fig. 1). Binding assays with purified *in vitro* phosphorylated Rad51 and Rad52, as well as coimmunoprecipitation of Rad51 and Rad52 from irradiated cells were performed. The results indicated that phosphorylation enhances the interaction between these two proteins *in vitro* and *in vivo*. The authors hypothesized that this irradiation-induced phosphorylation of Rad51 on tyrosine residues and the concomitant increase in association with Rad52 may lead to increased DNA repair efficiency (Chen et al., 1999b). *In vitro* studies with different Y315 mutants suggest that the phosphorylation of this residue is important for the binding of Rad51 to dsDNA and for nucleofilament formation (Takizawa et al., 2004). Moreover, Y315 is located near the polymerisation site of the protein, a region which is essential for the filament formation of Rad51 on DSBs, (Conilleau et al., 2004).

Slupianek and colleagues analyzed the role of Rad51 phosphorylation in the resistance of cells to DNA damaging agents. The resistance of BCR/Abl expressing cells to cisplatin and mitomycin C was decreased upon overexpression of nonphosphorylatable Rad51 Y315F. The mutation of Y54 had no effect on resistance. These results link the phosphorylation of Y315 to the resistance to DNA cross-linking agents and suggest that it has an important impact on DNA repair (Slupianek et al., 2001).

Recently, the same team reported an implication of Y315 phosphorylation in the regulation of BCR/Abl-Rad51 interaction. BCR/Abl-mediated phosphorylation of Y315 appears to be important for the dissociation of Rad51 from BCR/Abl in chronic myeloid leukemia cells (Slupianek et al., 2009). The authors studied the intracellular localization of wild type and mutated Rad51 in response to DSBs induced by genotoxic treatment. The nonphosphorylatable Rad51 Y315F mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/Abl-positive cells. This indicates that phospho-Y315 stimulates abundant nuclear localization of Rad51 on DSBs.

#### **2.3 Phosphorylation on Threonine 309 by Chk1**

Rad51 can also be phosphorylated on threonine. Sorensen and colleagues observed that a Chk1 signal is necessary for efficient homologous recombination. The inhibition of this kinase decreased the level of homologous recombination and of DNA DSB repair. The inhibition of Chk1 also impaired the formation of Rad51 foci which was not due to decreased Rad51 levels. The interaction of Rad51 with chromatin was dependent on Chk1 activity. Using immunoprecipitation, Sorensen and colleagues showed that Chk1 and Rad51 can interact physically in cells. Chk1 phosphorylates Rad51 on Threonine 309 which is located in a Chk1 consensus phosphorylation site. Cells transfected with a nonphosphorylatable Rad51 mutant were more sensitive to hydroxyurea which confirms that Chk1 signaling is required for homologous recombination repair (Sorensen et al., 2005).
