Rad52 Sumoylation Prevents the Toxicity of Unproductive Rad51 Filaments Independently of the Anti-Recombinase Srs2

Abstract

The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

“…2A and Ref. [78]). Taken together, these data indicate that physiological SUMOylation of Rad52 and Rad59 has no pronounced impact on survival after DNA damage.…”

“…Consistent with a previous report [44], the non-SUMOylatable Rad52-3KR and Rad59-2KR displayed reduced protein levels, while fusion to Rad59 increased its steady-state level, suggesting that SUMOylation may shield Rad52 and Rad59 against degradation. To investigate whether SUMOylation of Rad59 acts redundantly with Srs2 to prevent or revert toxic recombination intermediates [34,77,78], we studied cell survival in both SRS2 and srs2 Δ backgrounds. Finally, we compared the DNA damage sensitivity of haploid and diploid cells to test whether the presence of a homologous chromosome would impact on survival of the rad59 mutants.…”

“…Interestingly, in diploid cells, the Rad52-Smt3ΔGG fusion rescued the Zeocin sensitivity of srs2 Δ cells and Rad52-Smt3ΔGG together with Rad59-Smt3ΔGG rescued the MMS sensitivity. Since MMS treatment leads to replication stress and most known suppressors of srs2 Δ reduce Rad51 nucleoprotein filament formation [78,91,92], we propose that SUMOylation of Rad59 is important for restraining toxic Rad51-mediated recombination during replication stress. This conclusion goes well with other reports of a role for Rad59 in DNA replication stress tolerance [3,93,94].…”

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability

“…Consistently, GCRs also increase in slx5 mutants (Nagai et al 2008;Oza et al 2009), suggesting that a repair mechanism that is prone to creating genome rearrangements is at work in this background. Sumoylated Rad52 constitutes the minority of the Rad52 proteins in the cell (Altmannova et al 2010;Esta et al 2013); thus, degradation of only this form would still leave unmodified Rad52 available for mediating recombination. Sumoylated Rad52 exhibits reduced mediator activity in Rad51 filament formation compared with the nonsumoylated form (Esta et al 2013).…”

“…Sumoylated Rad52 constitutes the minority of the Rad52 proteins in the cell (Altmannova et al 2010;Esta et al 2013); thus, degradation of only this form would still leave unmodified Rad52 available for mediating recombination. Sumoylated Rad52 exhibits reduced mediator activity in Rad51 filament formation compared with the nonsumoylated form (Esta et al 2013). Conversely, the nonsumoylatable Rad52-3KR protein causes increased recombination within the rDNA and at direct and inverted repeats (Altmannova et al 2010).…”