**1. Introduction**

142 DNA Repair

[93] Field, S.J., et al., *E2F-1 functions in mice to promote apoptosis and suppress proliferation.* Cell,

[94] Wang, S., et al., *JWA regulates XRCC1 and functions as a novel base excision repair protein in* 

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1936-50

Double-strand breaks (DSB) are probably the most deleterious form of DNA alteration in a cell. They may arise from ionizing radiation, free radicals, chemicals, or during replication of single-strand breaks. There are two distinct and complementary mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homologous recombination (HR). Both repair pathways are important for the elimination of DSBs in eukaryotes.

Although the mechanisms of the cellular choice between these two pathways remain unclear, there is evidence that it depends on the cell cycle, as well as on mechanisms such as posttranslational modifications. When an intact DNA copy is available, HR is preferred and it is mainly active during late S and G2 phases of the cell cycle, while NHEJ is predominant during G0 and early S phases. The NHEJ pathway is characterised by a phosphorylation cascade where the first step is the activation of DNA-PKc protein which comprises a catalytic subunit and which is essential to complete the repair process. In contrast to NHEJ, the role of posttranslational modifications of proteins involved in the HR pathway is not clearly defined. Rad51 is a central protein in HR repair and its activity is based on pairing and strand exchange between homologous DNAs. The molecular regulation of Rad51 levels and activity has not been completely established. However, the kinase-induced phosphorylation of this protein modulates its recombinase activity by changing its interface and recognition sites and probably its intracellular distribution. Indeed, Rad51 associates with its paralogues and with other partner proteins, such as Rad52, Rad54, BRCA2 tumour suppressor, BLM helicase (Fig.1). Rad51 forms distinct subnuclear complexes called foci, which represent the functional units in DNA repair by HR. This accumulation of repair proteins to sites of double-strand break repair is closely dependant on protein-protein interactions which can be regulated by posttranslational modification processes including tyrosine, serine and threonine phosphorylations. This underlines the high complexity of HR regulation in mammalian cells.

Regulation of Rad51 recombinase activity and its interactions following DNA damage are poorly understood. In this chapter we have summarized the posttranslational modifications

<sup>\*</sup> M.P. and S.H. contributed equally to this work

Posttranslational Modifications of Rad51 Protein and Its Direct Partners:

**2.1.1 Phosphorylation on Tyrosine 54** 

Role and Effect on Homologous Recombination – Mediated DNA Repair 145

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

*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.,

Two years after Yuan and colleagues published their study, another group demonstrated that Rad51 can be phosphorylated. However Chen and colleagues did not observe the phosphorylation of Tyrosine 54 but detected the phosphorylation of another tyrosine

The authors used GST pull-down assays and immunoprecipitation to show that Rad51 forms a complex with c-Abl and ATM in cells. The association between the three proteins was independent of irradiation and DNA binding. The level of phosphorylation of Rad51 after irradiation of cells was investigated. The analyses of immunoprecipitated Rad51 with an anti-phosphoTyrosine antibody showed that the level of phosphorylation increases after irradiation. Rad51 was a direct substrate for c-Abl and the phosphorylation was dependent on both c-Abl and ATM. In order to determine which tyrosine residue was phosphorylated, the authors co-expressed c-Abl and wild type or mutated Rad51 in cells. Different tyrosine to phenylalanine Rad51 mutants were performed. Phenylalanine is an amino acid that cannot be phosphorylated. Thus, a signal would no longer be detected by the antiphosphoTyrosine antibody when the phosphorylated residue is mutated. The mutation of Y315 to phenylalanine abolished Rad51 phosphorylation, indicating that c-Abl

Rad51 can also be phosphorylated by the oncogenic fusion tyrosine kinase BCR/Abl. BCR/Abl is expressed in most cases of chronic myeloid leukemia and in some cases of acute

Slupianek and colleagues suggested that Rad51 and BCR/Abl interact physically since a portion of Rad51 co-localizes with the fusion tyrosine kinase in the cytoplasm of BCR/Abl overexpressing cells. This interaction was confirmed by the co-immunoprecipitation of the

Rad51 was immunoprecipitated from cells overexpressing BCR/Abl and its phosphorylation state was examined with an anti-phosphoTyrosine antibody. The interaction between

showed that the binding is direct and is mediated by the SH3 domain of c-Abl.

1999b; Chen et al., 1999c; Dong et al., 1999; Yuan et al., 1999; Zhong et al., 1999).

**2.1.2 Phosphorylation on Tyrosine 315 by c-Abl** 

phosphorylates Rad51 on this residue (Yuan et al., 1998).

**2.1.3 Phosphorylation on Tyrosine 315 by BCR/Abl** 

myeloid leukemia and possesses constitutive kinase activity.

residue, in position 315.

two proteins.

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 intracellular distribution of DNA repair proteins.

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