Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction

Abstract

Srs2 plays many roles in DNA repair, the proper regulation and coordination of which is essential. Post-translational modification by small ubiquitin-like modifier (SUMO) is one such possible mechanism. Here, we investigate the role of SUMO in Srs2 regulation and show that the SUMO-interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of SIM in a srs2⌬PIM strain leads to a decrease in recombination, indicating a pro-recombination role of SUMO. Thus SIM has an ambivalent function in Srs2 regulation; it not only mediates interaction with SUMO-PCNA to promote the anti-recombination function but it also plays a PCNA-independent pro-recombination role, probably by stimulating the formation of recombination complexes. The fact that deletion of PIM suppresses the phenotypes of Srs2 lacking SIM suggests that proper balance between the anti-recombination PCNA-bound and pro-recombination pools of Srs2 is crucial. Notably, sumoylation of Srs2 itself specifically stimulates recombination at the rDNA locus.

Multifunctional Roles of Saccharomyces cerevisiae Srs2 protein in Replication, Recombination and Repair

“…Regulation of Srs2 in this situation does not involve interaction with PCNA but is stimulated by SUMO and the SUMO-like protein Esc2 (Urulangodi et al 2015;Kolesar et al 2016). …”

“…In addition to the interaction with PCNA, a more recent study suggests that the SIM domain of Srs2 also mediates its interaction with other recombination factors, e.g. Mre11, Rad51 and Rad52, and likely helps to fulfill a prorecombination role (Kolesar et al 2016).…”

“…Of note is the srs2 rad54 double mutant lethality, which is due to inappropriate recombination. Since Rad54 protein is needed for many steps during HR, the double mutant lethality, which is rescued by rad51 mutation, suggests that Srs2 has a prorecombination role that overlaps with Rad54 (Klein 2001;Ira et al 2003;Kolesar et al 2016). In some cases, lethality is not rescued by RAD51 deletion as recombination repair is essential to collapsed replication fork restart (Debrauwere et al 2001).…”

“…Srs2 regulates repair at stalled replication forks in part though recruitment to SUMO-modified PCNA (Papouli et al 2005;Pfander et al 2005); however, Srs2 can remove lethal recombination intermediates independent of this interaction Kolesar et al 2016). In addition to regulating recombination at stalled forks, the Srs2 protein regulates recombination by limiting crossovers and promoting SDSA (Ira et al 2003;Robert et al 2006;Miura, Shibata and Kusano 2013), thus limiting loss of heterozygosity events during mitotic repair of lesions through HR.…”

DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae

“…Importantly, Srs2 was also shown to have the ability to dismantle Rad51 nucleofilaments and to inhibit Rad51-driven DNA strand exchange (Krejci et al 2003;Veaute et al 2003). The Srs2 helicase itself can undergo SUMOylation, which disfavors its binding to SUMO-PCNA but is important for its interaction with Rad51 and other recombination factors (Kolesar et al 2012(Kolesar et al , 2016. Therefore, modulation of the equilibrium between unmodified and modified forms of Srs2 plays a role in the choice of pathway used to deal with replication stress.…”