title: The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair.
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 Fml1(K99R) and Fml1(D196N) are proficient for DNA binding but totally deficient in DNA unwinding and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1Δ mutant indicating that Fml1's motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intriguingly, both fml1(K99R) and fml1(D196N) mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1Δ strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA substrate can impede alternative mechanisms of repair and crossover avoidance.
authors: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
authors: Saikat
authors: S
authors: Nandi
authors: Matthew C
authors: MC
authors: Whitby
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D045566:DNA, Cruciform / Q000378:metabolism; D005785:Gene Conversion; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008938:Mitosis / Q000235:genetics; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D059767:Recombinational DNA Repair; D012568:Schizosaccharomyces / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D045566:DNA, Cruciform; D009153:Mutagens; D029702:Schizosaccharomyces pombe Proteins; D004247:DNA; D008741:Methyl Methanesulfonate; D000251:Adenosine Triphosphatases; C573663:Fml1 protein, S pombe; D004265:DNA Helicases