**2.4 Sumoylation – Ubiquitination of Rad51**

148 DNA Repair

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

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,

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

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

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

phospho-Y315 stimulates abundant nuclear localization of Rad51 on DSBs.

to ssDNA, phosphorylation inhibits Rad51 function (Yuan et al., 1998).

(Conilleau et al., 2004).

et al., 2005).

impact on DNA repair (Slupianek et al., 2001).

**2.3 Phosphorylation on Threonine 309 by Chk1** 

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 damage (Saitoh et al., 2002).

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