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.

Department of Biology National Centre for Biomolecular Research Masaryk University 62500 Brno Czech Republic

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Li X, Heyer W-D. Homologous recombination in DNA repair and DNA damage tolerance. Cell Res. 2008;18:99–113. PubMed PMC

Budzowska M, Kanaar R. Mechanisms of dealing with DNA damage-induced replication problems. Cell Biochem. Biophys. 2009;53:17–31. PubMed

Reliene R, Bishop AJR, Schiestl RH. Involvement of homologous recombination in carcinogenesis. Adv. Genet. 2007;58:67–87. PubMed

Krejci L, Altmannova V, Spirek M, Zhao X. Homologous recombination and its regulation. Nucleic Acids Res. 2012;40:5795–5818. PubMed PMC

Marini V, Krejci L. Srs2: the “odd-job man” in DNA repair. DNA Repair (Amst.) 2010;9:268–275. PubMed PMC

Fugger K, Mistrik M, Danielsen JR, Dinant C, Falck J, Bartek J, Lukas J, Mailand N. Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities. J. Cell Biol. 2009;186:655–663. PubMed PMC

Moldovan G-L, Dejsuphong D, Petalcorin MIR, Hofmann K, Takeda S, Boulton SJ, D’Andrea AD. Inhibition of homologous recombination by the PCNA-interacting protein PARI. Mol. Cell. 2012;45:75–86. PubMed PMC

Lawrence CW, Christensen RB. Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants. J. Bacteriol. 1979;139:866–876. PubMed PMC

Schiestl RH, Prakash S, Prakash L. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway. Genetics. 1990;124:817–831. PubMed PMC

Aguilera A, Klein HL. Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics. 1988;119:779–790. PubMed PMC

Fabre F, Chan A, Heyer W-D, Gangloff S. Alternate pathways involving Sgs1/Top3, Mus81/Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc. Natl. Acad. Sci. U.S.A. 2002;99:16887–16892. PubMed PMC

Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, Klein H, Ellenberger T, Sung P. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature. 2003;423:305–309. PubMed

Veaute X, Jeusset J, Soustelle C, Kowalczykowski SC, Le Cam E, Fabre F. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature. 2003;423:309–312. PubMed

Pfander B, Moldovan G-L, Sacher M, Hoege C, Jentsch S. SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature. 2005;436:428–433. PubMed

Papouli E, Chen S, Davies AA, Huttner D, Krejci L, Sung P, Ulrich HD. Crosstalk between SUMO and ubiquitin on PCNA is mediated by recruitment of the helicase Srs2p. Mol. Cell. 2005;19:123–133. PubMed

Burgess RC, Lisby M, Altmannova V, Krejci L, Sung P, Rothstein R. Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo. J. Cell Biol. 2009;185:969–981. PubMed PMC

Anand RP, Shah KA, Niu H, Sung P, Mirkin SM, Freudenreich CH. Overcoming natural replication barriers: differential helicase requirements. Nucleic Acids Res. 2012;40:1091–1105. PubMed PMC

Kerrest A, Anand RP, Sundararajan R, Bermejo R, Liberi G, Dujon B, Freudenreich CH, Richard G-F. SRS2 and SGS1 prevent chromosomal breaks and stabilize triplet repeats by restraining recombination. Nat. Struct. Mol. Biol. 2009;16:159–167. PubMed PMC

Dhar A, Lahue RS. Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae. Nucleic Acids Res. 2008;36:3366–3373. PubMed PMC

Bhattacharyya S, Lahue RS. Saccharomyces cerevisiae Srs2 DNA helicase selectively blocks expansions of trinucleotide repeats. Mol. Cell. Biol. 2004;24:7324–7330. PubMed PMC

León Ortiz AM, Reid RJD, Dittmar JC, Rothstein R, Nicolas A. Srs2 overexpression reveals a helicase-independent role at replication forks that requires diverse cell functions. DNA Repair (Amst.) 2011;10:506–517. PubMed PMC

Ira G, Malkova A, Liberi G, Foiani M, Haber JE. Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell. 2003;115:401–411. PubMed PMC

Saponaro M, Callahan D, Zheng X, Krejci L, Haber JE, Klein HL, Liberi G. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair. PLoS Genet. 2010;6:e1000858. PubMed PMC

Sampson DA, Wang M, Matunis MJ. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J. Biol. Chem. 2001;276:21664–21669. PubMed

Bernier-Villamor V, Sampson DA, Matunis MJ, Lima CD. Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell. 2002;108:345–356. PubMed

Tatham MH, Chen Y, Hay RT. Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex. Biochemistry. 2003;42:3168–3179. PubMed

Takahashi H, Hatakeyama S, Saitoh H, Nakayama KI. Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein. J. Biol. Chem. 2005;280:5611–5621. PubMed

Lin D-Y, Huang Y-S, Jeng J-C, Kuo H-Y, Chang C-C, Chao T-T, Ho C-C, Chen Y-C, Lin T-P, Fang H-I, et al. Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol. Cell. 2006;24:341–354. PubMed

Meulmeester E, Kunze M, Hsiao HH, Urlaub H, Melchior F. Mechanism and consequences for paralog-specific sumoylation of ubiquitin-specific protease 25. Mol. Cell. 2008;30:610–619. PubMed

Knipscheer P, Flotho A, Klug H, Olsen JV, van Dijk WJ, Fish A, Johnson ES, Mann M, Sixma TK, Pichler A. Ubc9 sumoylation regulates SUMO target discrimination. Mol. Cell. 2008;31:371–382. PubMed

Zhu J, Zhu S, Guzzo CM, Ellis NA, Sung KS, Choi CY, Matunis MJ. Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J. Biol. Chem. 2008;283:29405–29415. PubMed PMC

Johnson ES, Gupta AA. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell. 2001;106:735–744. PubMed

Takahashi Y, Toh-e A, Kikuchi Y. A novel factor required for the SUMO1/Smt3 conjugation of yeast septins. Gene. 2001;275:223–231. PubMed

Zhao X, Blobel G. A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc. Natl. Acad. Sci. U.S.A. 2005;102:4777–4782. PubMed PMC

Erdeniz N, Mortensen UH, Rothstein R. Cloning-free PCR-based allele replacement methods. Genome Res. 1997;7:1174–1183. PubMed PMC

Colavito S, Macris-Kiss M, Seong C, Gleeson O, Greene EC, Klein HL, Krejci L, Sung P. Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption. Nucleic Acids Res. 2009;37:6754–6764. PubMed PMC

Bencsath KP, Podgorski MS, Pagala VR, Slaughter CA, Schulman BA. Identification of a multifunctional binding site on Ubc9p required for Smt3p conjugation. J. Biol. Chem. 2002;277:47938–47945. PubMed

Johnson ES, Blobel G. Ubc9p is the conjugating enzyme for the ubiquitin-like protein Smt3p. J. Biol. Chem. 1997;272:26799–26802. PubMed

Johnson ES, Schwienhorst I, Dohmen RJ, Blobel G. The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer. EMBO J. 1997;16:5509–5519. PubMed PMC

Takahashi Y, Kahyo T, Toh-E A, Yasuda H, Kikuchi Y. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions as an adaptor between conjugating enzyme and substrates. J. Biol. Chem. 2001;276:48973–48977. PubMed

Takahashi Y, Toh-E A, Kikuchi Y. Comparative analysis of yeast PIAS-type SUMO ligases in vivo and in vitro. J. Biochem. 2003;133:415–422. PubMed

Haracska L, Unk I, Prakash L, Prakash S. Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 2006;103:6477–6482. PubMed PMC

Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo N, Jiang L, Piccirillo S, Yu H, et al. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 2005;19:2816–2826. PubMed PMC

Marini V, Krejci L. Unwinding of replication and recombination substrates by Srs2. DNA Repair. 2012 in press. PubMed PMC

Altmannova V, Eckert-Boulet N, Arneric M, Kolesar P, Chaloupkova R, Damborsky J, Sung P, Zhao X, Lisby M, Krejci L. Rad52 SUMOylation affects the efficiency of the DNA repair. Nucleic Acids Res. 2010;38:4708–4721. PubMed PMC

Krejci L, Damborsky J, Thomsen B, Duno M, Bendixen C. Molecular dissection of interactions between Rad51 and members of the recombination-repair group. Mol. Cell. Biol. 2001;21:966–976. PubMed PMC

Hang LE, Liu X, Cheung I, Yang Y, Zhao X. SUMOylation regulates telomere length homeostasis by targeting Cdc13. Nat. Struct. Mol. Biol. 2011;18:920–926. PubMed PMC

Ho C-W, Chen H-T, Hwang J. UBC9 autosumoylation negatively regulates sumoylation of septins in Saccharomyces cerevisiae. J. Biol. Chem. 2011;286:21826–21834. PubMed PMC

Dou H, Huang C, Van Nguyen T, Lu L-S, Yeh ETH. SUMOylation and de-SUMOylation in response to DNA damage. FEBS Lett. 2011;585:2891–2896. PubMed

Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature. 2009;458:461–467. PubMed

Ulrich HD. The SUMO system: an overview. Methods Mol. Biol. 2009;497:3–16. PubMed

Cremona CA, Sarangi P, Yang Y, Hang LE, Rahman S, Zhao X. Extensive DNA damage-induced sumoylation contributes to replication and repair and acts in addition to the Mec1 checkpoint. Mol. Cell. 2012;10 1016/j.molcel.2011.11.028. PubMed PMC

Burgess RC, Rahman S, Lisby M, Rothstein R, Zhao X. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Mol. Cell. Biol. 2007;27:6153–6162. PubMed PMC

Yunus AA, Lima CD. Structure of the Siz/PIAS SUMO E3 ligase Siz1 and determinants required for SUMO modification of PCNA. Mol. Cell. 2009;35:669–682. PubMed PMC

Duan X, Sarangi P, Liu X, Rangi GK, Zhao X, Ye H. Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex. Mol. Cell. 2009;35:657–668. PubMed PMC

Johnson ES. Protein modification by SUMO. Annu. Rev. Biochem. 2004;73:355–382. PubMed

Reindle A, Belichenko I, Bylebyl GR, Chen XL, Gandhi N, Johnson ES. Multiple domains in Siz SUMO ligases contribute to substrate selectivity. J. Cell. Sci. 2006;119:4749–4757. PubMed

Baba D, Maita N, Jee J-G, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M. Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature. 2005;435:979–982. PubMed

Steinacher R, Schär P. Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation. Curr. Biol. 2005;15:616–623. PubMed

Hoeller D, Crosetto N, Blagoev B, Raiborg C, Tikkanen R, Wagner S, Kowanetz K, Breitling R, Mann M, Stenmark H, et al. Regulation of ubiquitin-binding proteins by monoubiquitination. Nat. Cell Biol. 2006;8:163–169. PubMed

Shen TH, Lin H-K, Scaglioni PP, Yung TM, Pandolfi PP. The mechanisms of PML-nuclear body formation. Mol. Cell. 2006;24:331–339. PubMed PMC

Armstrong AA, Mohideen F, Lima CD. Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature. 2012;483:59–63. PubMed PMC

Hecker C-M, Rabiller M, Haglund K, Bayer P, Dikic I. Specification of SUMO1- and SUMO2-interacting motifs. J. Biol. Chem. 2006;281:16117–16127. PubMed

Song J, Durrin LK, Wilkinson TA, Krontiris TG, Chen Y. Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl. Acad. Sci. U.S.A. 2004;101:14373–14378. PubMed PMC

Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 2007;8: 947–956. PubMed

Hoege C, Pfander B, Moldovan G-L, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002;419:135–141. PubMed

Leach CA, Michael WM. Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts. J. Cell Biol. 2005;171: 947–954. PubMed PMC

Arakawa H, Moldovan G-L, Saribasak H, Saribasak NN, Jentsch S, Buerstedde J-M. A role for PCNA ubiquitination in immunoglobulin hypermutation. PLoS Biol. 2006;4:e366. PubMed PMC

Le Breton C, Dupaigne P, Robert T, Le Cam E, Gangloff S, Fabre F, Veaute X. Srs2 removes deadly recombination intermediates independently of its interaction with SUMO-modified PCNA. Nucleic Acids Res. 2008;36:4964–4974. PubMed PMC

Thomas BJ, Rothstein R. The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics. 1989;123:725–738. PubMed PMC

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