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 (Rad51E108L) was predicted to bind more strongly with Hsp90 than the wild-type (Rad51WT). A coimmunoprecipitation study demonstrated that there exists a distinct difference between the in vivo associations of Rad51WT-Hsp90 and of Rad51E108L-Hsp90. We found that upon DNA damage, the association between Rad51WT and Hsp90 was significantly reduced compared to that in the undamaged condition. However, the mutant Rad51E108L remained tightly associated with Hsp90 even after DNA damage. Consequently, the recruitment of Rad51E108L to the double-stranded broken ends was reduced significantly. The E108L-rad51 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 Rad51E108L 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. IMPORTANCE Rad51-mediated homologous recombination is the major mechanism for repairing DNA double-strand break (DSB) repair in cancer cells. Thus, regulating Rad51 activity could be an attractive target. The sequential assembly and disassembly of Rad51 to the broken DNA ends depend on reversible protein-protein interactions. Here, we discovered that a dynamic interaction with molecular chaperone Hsp90 is one such regulatory event that governs the recruitment of Rad51 onto the damaged DNA. We uncovered that Rad51 associates with Hsp90, and upon DNA damage, this complex dissociates to facilitate the loading of Rad51 onto broken DNA. In a mutant where such dissociation is incomplete, the occupancy of Rad51 at the broken DNA is partial, which results in inefficient DNA repair. Thus, it is reasonable to propose that any small molecule that may alter the dynamics of the Rad51-Hsp90 interaction is likely to impact DSB repair in cancer cells.

DNA damage-induced nuclear import of HSP90α is promoted by Aha1

“…We investigated whether the nuclear translocation of yHSP90α occurs in an aforementioned phosphorylation-independent manner in S. cerevisiae , in which the first 14 amino acids are absent (Supplemental Figure S1, red circle). Earlier we observed that the association between Rad51 and yHSP90α was reduced in the presence of MMS (0.15%); hence we used the same dose of MMS for our present study ( Suhane et al. , 2019 ).…”

“…A half batch of cells was treated with 0.15% of MMS and grown at 30°C for 2 h along with the untreated batch of cells. Cells were harvested and the pull down was done by using anti-FLAG antibody as described previously ( Suhane et al. , 2019 ).…”

“…Studies in the model eukaryote, Saccharomyces cerevisiae , have shown that yHSP90α, the budding yeast ortholog of human HSP90α (HsHSP90α), provides stability to Rad51 protein and plays a regulatory role in the effective recruitment of Rad51 to the broken DNA ( Suhane et al. , 2014 , 2019 ).…”

Identification of a chaperone-code responsible for Rad51-mediated genome repair

“…CoIP was performed using the previously used protocol ( 13 ). Briefly, KRAY20, KRAY3, KRAY4, KRAY5, KRAY6 , NFY24, KRAY54, KRAY55, KRAY56 , and KRAY57 strains were grown in YPD medium at A 600 till 0.5 at 30 °C.…”

“…Furthermore, it was reported that the strain harboring an ATPase dead mutant of Hsp82, experiences extreme sensitization to DNA mutagenic agents along with the destabilization of Rad51 and Rad52 ( 12 ). It was observed that although deleting the charged linker region of Hsp82 does not hamper Rad51 stability, but it reduces methyl methane sulfonate (MMS)-dependent Rad51 nuclear foci formation, and impairs Rad51 activity during HR ( 12 , 13 ). Although it is known that Hsp82 regulates the stability and activity of Rad51, but the comprehensive understanding of the mechanism behind this regulation remains obscure.…”