**2.1.1 Phosphorylation on Tyrosine 54**

144 DNA Repair

of Rad51 and of the proteins interacting physically with Rad51 during HR repair. We then attempt to relate the impact of these modifications on HR DNA repair and on the

Fig. 1. Schematic representation of the mechanism of DNA DSB repair by homologous

**2.1 Tyrosine phosphorylation of Rad51 by the c-Abl family of tyrosine kinases** 

different site than Tyrosine 315 in MEF cAbl-/- cells (Chen et al., 1999b).

Several studies have shown that Rad51 can be phosphorylated on tyrosine but until recently there were discrepancies on the exact site of phosphorylation. Three studies had shown the phosphorylation of Tyrosine 315 (Y315) and only one the phosphorylation of Tyrosine 54 (Y54). A recent publication demonstrated that both of these tyrosines can be phosphorylated. The kinases which phosphorylate Rad51 belong to the c-Abl family which has two members, c-Abl and Arg. The oncogenic fusion tyrosine kinase BCR/Abl has also been shown to phosphorylate Rad51. However, other tyrosine kinases can also phosphorylate Rad51 at a

**2. Post-translational modifications of Rad51** 

intracellular distribution of DNA repair proteins.

recombination.

The first study showing that Rad51 can be phosphorylated was published in 1998 by Yuan and colleagues. Using co-immunoprecipitation, the authors observed that human Rad51 (hRad51) binds to c-Abl in cells. This association was unaffected by irradiation of the cells and was not dependent on DNA binding. Pull-down assays were performed with a GST-c-Abl fusion protein or a GST-c-Abl SH3 domain fusion peptide. These were incubated with cell lysates or purified hRad51. The results confirmed the association between hRad51 and c-Abl *in vitro* and showed that the binding is direct and is mediated by the SH3 domain of c-Abl.

*In vitro* phosphorylation assays with purified c-Abl and hRad51 demonstrated that hRad51 is a substrate for this kinase. Immunoprecipitation of Rad51 was performed with lysates from irradiated cells overexpressing hRad51 and c-Abl. The analyses of the immunoprecipitated protein with an anti-phosphoTyrosine antibody confirmed the phosphorylation of Rad51 *in vivo*. The *in vivo* and *in vitro* phosphorylated hRad51 proteins were then purified and analyzed by mass spectroscopy. The detected peaks indicated that the phosphorylation is located on Tyrosine 54 on both *in vivo* and *in vitro* phosphorylated Rad51 (Chen et al., 1999a; Chen et al., 1999b; Chen et al., 1999c; Dong et al., 1999; Yuan et al., 1999; Zhong et al., 1999).
