4964–4974 Nucleic Acids Research, 2008, Vol. 36, No. 15 doi:10.1093/nar/gkn441 Published online 25 July 2008 Srs2 removes deadly recombination intermediates independently of its interaction with SUMO-modified PCNA Cyrille Le Breton 1 , Pauline Dupaigne 2 , Thomas Robert1, Eric Le Cam2, Serge Gangloff1, Francis Fabre1 and Xavier Veaute1,* 1CEA–DSV-Institut de Radiobiologie Cellulaire et Mole´ culaire, UMR217 CNRS/CEA, F-92265 Fontenay aux Roses and 2Laboratoire de Microscopie Mole´ culaire et Cellulaire, UMR 8126, Interactions Mole´ culaires et Cancer CNRS-Universite´ Paris Sud-Institut de Cance´ rologie Gustave Roussy, F-94805 Villejuif, France Received December 18, 2007; Revised June 3, 2008; Accepted June 26, 2008 ABSTRACT Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair defi- cient cells, and a second one is to eliminate poten- tially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon transloca- tion on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar pheno- types, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to dis- place Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed. INTRODUCTION DNA helicases are present in all kingdoms and perform an extraordinary variety of functions in cells. They play important roles in DNA metabolic processes such as repli- cation, repair, recombination and transcription. Their DNA unwinding and translocase activities begin to be well characterized at the biochemical and structural levels (1). However, much remains to be understood about their roles in vivo. Since all organisms express multiple DNA helicases, a high degree of functional specialization should exist. This is particularly true for the control of homologous recombination (HR) (2). In Saccharomyces cerevisiae, at least six diﬀerent helicases, Sgs1, Srs2, Mph1, Mer3, Pif1 and Rrm3 are positively or negatively involved in HR, increasing or decreasing recombination rates. They all perform speciﬁc functions that cannot be executed by another one. When and how these helicases act remains unclear. Partial answers to these questions have been recently obtained for Srs2. Srs2 belongs to the SF1 helicase family and is structu- rally and functionally related to the bacterial UvrD heli- case (3,4). Mutants of SRS2 were ﬁrst described as partial suppressors of the radiation sensitivity of rad6 and rad18 mutants (5), defective in postreplicative DNA repair (PRR). They were later isolated as hyper-recombination mutants (6) and as suppressors of the methyl methane- sulfonate (MMS) sensitivity of rad18 cells (3). In RAD18 background, the single srs2 mutants are sensitive to ultraviolet (UV) light (3), a phenotype dependent on *To whom correspondence should be addressed. Tel: +33 1 46 54 93 43; Fax: +33 1 46 54 88 59; Email: xavier.veaute@cea.fr Present address: Thomas Robert, FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy The author wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.