Glu- 108 in Saccharomyces cerevisiae Rad51 is critical for DNA damage-induced nuclear function

Abstract

DNA damage-induced Rad51 focus formation is the hallmark of homologous recombination-mediated DNA repair. Earlier, we reported that Rad51 physically interacts with Hsp90, and under the condition of Hsp90 inhibition, it undergoes proteasomal degradation. Here, we show that the dynamic interaction between Rad51 and Hsp90 is crucial for the DNA damage-induced nuclear function of Rad51. Guided by a bioinformatics study, we generated a single mutant of Rad51, which resides at the N-terminal domain, outside the ATPase core domain. The mutant with an E to L change at residue 108 (Rad51 E108L ) was predicted to bind more strongly with Hsp90 than the wild-type (Rad51 WT ). A coimmunoprecipitation study demonstrated that there exists a distinct difference between the in vivo associations of Rad51WT-Hsp90 and of Rad51 E108L -Hsp90. We found that upon DNA damage, the association between Rad51 WT and Hsp90 was significantly reduced compared to that in the undamaged condition. However, the mutant Rad51 E108L remained tightly associated with Hsp90 even after DNA damage. Consequently, the recruitment of Rad51 E108L to the double-stranded broken ends was reduced significantly. The E108Lrad51 strain manifested severe sensitivity toward methyl methanesulfonate (MMS) and a complete loss of gene conversion efficiency, a phenotype similar to that of the Δrad51 strain. Previously, some of the N-terminal domain mutants of Rad51 were identified in a screen for a Rad51 interaction-deficient mutant; however, our study shows that Rad51 E108L is not defective either in the self-interaction or its interaction with the members of the Rad52 epistatic group. Our study thus identifies a novel mutant of Rad51 which, owing to its greater association with Hsp90, exhibits a severe DNA repair defect.