A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

Department of Biology Masaryk University Kamenice 5 A7 Brno 625 00 Czech Republic

Department of Biology Masaryk University Kamenice 5 A7 Brno 625 00 Czech Republic Department of Biology University of Copenhagen Ole Maaløes Vej 5 DK 2200 Copenhagen N Denmark

Department of Biology Masaryk University Kamenice 5 A7 Brno 625 00 Czech Republic International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno Brno Czech Republic

Department of Biology Masaryk University Kamenice 5 A7 Brno 625 00 Czech Republic National Centre for Biomolecular Research Masaryk University Kamenice 5 A4 Brno 625 00 Czech Republic

Department of Biology Masaryk University Kamenice 5 A7 Brno 625 00 Czech Republic National Centre for Biomolecular Research Masaryk University Kamenice 5 A4 Brno 625 00 Czech Republic International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno Brno Czech Republic

Department of Genetics Cancer Research Institute Vlarska 7 833 91 Bratislava Slovakia

Department of Molecular Biology and Biochemistry Rutgers University Piscataway NJ USA

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RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links

Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments

Esc2 promotes Mus81 complex-activity via its SUMO-like and DNA binding domains

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