Dual roles of the SUMO-interacting motif in the regulation of Srs2

Department of Biology, National Centre for Biomolecular Research, Masaryk University, 62500 Brno, Czech Republic, Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, Programs in Biochemistry, Cell, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA and International Clinical Research Center, Center for Biomolecular and Cellular Engineering, St. Anne's University Hospital in Brno, 60200 Brno, Czech Republic.
Nucleic Acids Research (Impact Factor: 9.11). 06/2012; 40(16):7831-43. DOI: 10.1093/nar/gks484
Source: PubMed


The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation-sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.

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    • "The SUMO targeted ubiquitin ligase (STUbL) Slx5–Slx8 complex plays a role in genome stability by controlling the turnover of SUMOylated factors in response to DNA damage (Sriramachandran and Dohmen 2014). As Esc2 and its Schizosaccharomyces pombe ortholog, Rad60, genetically and physically interact with Slx5–Slx8 (Prudden et al. 2007; Sollier et al. 2009) and as Srs2 is SUMOylated (Saponaro et al. 2010; Kolesar et al. 2012), we examined whether Srs2 degradation is also mediated by Slx5–Slx8. We found that Srs2 protein levels were stabilized following genotoxic stress in the absence of Slx5, similar to what we observed in esc2Δ (Fig. 6F). "
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    ABSTRACT: Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to rep-lication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recom-binase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.
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    • "In summary, our pull-down experiments suggest that Rad54 binds to the IDCL domain in PCNA, however, the putative PIP-box in Rad54 plays only a minor role in the interaction. Therefore, it is possible that Rad54 binds PCNA through an alternative, yet to be determined domain, such as the PIM (PCNA-interaction motif), which also binds to IDCL domain on PCNA [45], [46], or APIM (AlkB homologue 2 PCNA-interaction motif) [36]. "
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    ABSTRACT: Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.
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    • "Compared with the 0 hr of mitosis time point, an approximate fivefold increase in Srs2 was seen at 4–8 hr of meiosis. Overexpressed Srs2 generated a ladder on Western blotting, suggesting multiple post-translational modifications, e.g., SUMOlyation (Kolesar et al. 2012). As reported previously (Palladino and Klein 1992; Sasanuma et al. 2013), the srs2 deletion mutant exhibits reduced spore viability (36.8%), which indicates a critical role for this helicase in meiosis (Figure 1D). "
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    ABSTRACT: Homologous recombination is associated with the dynamic assembly and disassembly of DNA-protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Over-expression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 over-expression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.
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