9584–9595 Nucleic Acids Research, 2012, Vol. 40, No. 19 doi:10.1093/nar/gks715 Published online 27 July 2012 The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair Saikat Nandi and Matthew C. Whitby* Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK Received February 9, 2012; Revised June 18, 2012; Accepted July 3, 2012 ABSTRACT In fission yeast, the DNA helicase Fml1, which is an orthologue of human FANCM, is a key component of the machinery that drives and governs homologous recombination (HR). During the repair of DNA double-strand breaks by HR, it limits the occurrence of potentially deleterious crossover recombinants, whereas at stalled replication forks, it promotes HR to aid their recovery. Here, we have mutated conserved residues in Fml1’s Walker A (K99R) and Walker B (D196N) motifs to determine whether its activities are dependent on its ability to hydrolyse ATP. Both Fml1K99R and Fml1D196N are proficient for DNA binding but totally deficient in DNA unwind- ing and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1" mutant indicating that Fml1’s motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intri- guingly, both fml1K99R and fml1D196N mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1" strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA sub- strate can impede alternative mechanisms of repair and crossover avoidance. INTRODUCTION The FANCM family of DNA helicases/translocases plays key roles in homologous recombination (HR) and DNA repair in archaea and eukaryotes (1). Human FANCM is a component of a network of DNA repair proteins, which if mutated can result in the genetic disease Fanconi anaemia (FA) characterized by progressive bone marrow failure, various developmental abnormalities and cancer predisposition (2). A distinguishing feature of FA is cellular hypersensitivity to agents that cause DNA interstrand crosslinks (ICLs) such as mitomycin C and cisplatin indicating an underlying defect in the repair of this class of lesion (3–5). A key role for FANCM in ICL repair appears to be to promote the recruitment of the FA core complex (consisting of FANCA, B, C, E, F, G and L) to replication forks that are stalled at ICLs (6,7). The core complex then promotes the monoubiquitination of FANCD2 and FANCI, which is thought to activate them for the co-ordination of downstream repair pathways including HR and translesion DNA synthesis (TLS) (3–5). However, a series of genetic studies have shown that FANCM’s role in the repair/tolerance of DNA damage is not restricted to an involvement with the FA core complex. Additional roles include promoting the S-phase checkpoint (8–10), possibly by acting as a sensor of stalled/broken replication forks and/or regulating the loading of RPA, and limiting sister chro- matid exchange (11–13), which indicates that it has a role in controlling HR. At least some of these FA core complex-independent roles are conserved in lower eukary- otes. For example, yeast orthologues of FANCM (Mph1 in Saccharomyces cerevisiae and Fml1 in Schizosaccharomyces pombe) play a prominent role in limiting crossover recombinants during mitotic DNA double-strand break (DSB) repair, and in the case of Fml1, there is good evidence for its involvement in pro- cessing stalled replication forks (14,15). The FANCM family are Superfamily 2 (SF2) helicases/ translocases and as such are characterized by seven conserved motifs including a Walker A motif (motif I) that binds the triphosphate tail of ATP and a DEAH box (motif II), which is a version of the Walker B motif that binds magnesium ions and consequently plays an es- sential role in catalysing ATP hydrolysis (1). In vitro studies of puriﬁed FANCM family proteins have provided insight into how their various in vivo functions might be performed. A general ability to unwind and/or branch migrate DNA junctions that resemble intermedi- ates of HR and stalled replication forks has been noted. In particular, FANCM family members are proﬁcient at branch migrating Holliday junctions (HJs) and unwinding *To whom correspondence should be addressed. Tel: +44 1865 613239; Fax: +44 1865 613343; Email: matthew.whitby@bioch.ox.ac.uk The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.