Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

Department of Biochemistry and Molecular Pharmacology New York University School of Medicine New York New York

Institute of Molecular Genetics Academy of Sciences of the Czech Republic 14300 Prague Czech Republic

Institute of Molecular Genetics Academy of Sciences of the Czech Republic 14300 Prague Czech Republic; Institute of Molecular Cancer Research University of Zurich CH 8057 Zurich Switzerland

Nordea Center for Healthy Aging Department of Cellular and Molecular Medicine University of Copenhagen 2200 Copenhagen N Denmark

Weatherall Institute of Molecular Medicine Department of Oncology University of Oxford Oxford OX3 9DS United Kingdom

Weatherall Institute of Molecular Medicine Department of Oncology University of Oxford Oxford OX3 9DS United Kingdom; Nordea Center for Healthy Aging Department of Cellular and Molecular Medicine University of Copenhagen 2200 Copenhagen N Denmark

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Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Human RECQ5 helicase promotes repair of DNA double-strand breaks by synthesis-dependent strand annealing