Abstract
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.
Highlights
Homologous recombination is regulated both positively and negatively in eukaryotic cells to suppress genomic instability
The F-box helicase 1 (FBH1) helicase has been implicated in the regulation of the RAD51 recombinase in a number of organisms, including fission yeast, as well as chicken and mammalian cells (14 –19)
Using biochemical and biophysical techniques, we have demonstrated that human FBH1 has the capacity to disrupt the ATP-bound form of the RAD51 presynaptic filament by acting as a ssDNA translocase
Summary
Homologous recombination is regulated both positively and negatively in eukaryotic cells to suppress genomic instability. A pivotal player in HR is the RAD51 protein (RecA in bacteria), a DNAdependent ATPase that polymerizes on ssDNA to form a nucleoprotein filament This structure is a central intermediate in HR and is necessary for DNA strand invasion to occur as part of the search for a homologous DNA sequence [1, 3]. A recently identified family member in human cells is PARI [11] This protein binds to RAD51, but it contains amino acid substitutions in critical residues required for ATPase function, and PARI is not an active helicase/translocase. Fbh limits Rad51-dependent recombination at blocked replication forks in a manner dependent on its helicase/translocase activity [16] Taken together, these findings suggest that Fbh regulates HR through directly regulating Rad function, at least in S. pombe. Our data provide mechanistic insight into the role of FBH1 as a negative regulator of RAD51 function in order to prevent unwanted HR
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