The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

Abstract

Homologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. The Saccharomyces cerevisiae yeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a new SRS2 allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of the srs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.

Rad51 filament dynamics and its antagonistic modulators

“…Sequence analysis of Srs2 has found a putative BRC repeat (F**A motif) and mutation of the key phenylalanine to an alanine residue (F891A) results in complete disruption of Rad51 interaction, but no change in sensitivity to the DNA damaging agent Methyl methanesulfonate (MMS) or overall cell viability [126]. This observation is also supported by the finding that removing the Rad51-binding site in Srs2 results in no observable differences to cell viability (as measured by cell survival over time) or response to genotoxic stress [127,128].…”

Role of the Srs2–Rad51 Interaction Domain in Crossover Control in Saccharomyces cerevisiae

“…The apparent discrepancy between the in vivo and in vitro roles of the Srs2–Rad51 interaction may be because in vivo there are other factors, such as PCNA, that are equally, if not more, important for the recruitment of Srs2 for Rad51 filament disruption. In fact, a recent genetic study on SRS2 concluded that the main role of Srs2 in DNA repair depends on its helicase/translocase activity instead of its Rad51or PCNA interaction (Bronstein et al 2018).…”

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae

“…Of note, the inhibition of repair by Srs2 is not likely to be by Rad51 eviction, since OE of Rad51, in contrast to that of Rad52, has no suppression effect. Instead, we assume that Srs2 directly inhibits, or counteracts, the activity of Rad52 (21,22). In support of this idea, a physical interaction was characterized between Srs2 and Rad52 (47).…”

“…PCNA SUMOylation at K164 seems to be evolutionarily conserved and has been observed also in other organisms (17,18). SUMOylation of PCNA can lead to the recruitment of Srs2, a UvrD-like helicase that can disrupt Rad51 presynaptic filaments and thus prevent homologous recombination (HR) (19)(20)(21)(22)(23). Functional homologs of Srs2 seem to exist in other organisms (e.g., PARI [18] and RTEL1 [24]).…”