Abstract
Srs2 is a helicase and single-stranded (ss)DNA translocase that can dismantle presynaptic Rad51 filaments in its capacity as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes this remains unclear. Here we report a single molecule fluorescence study of the dynamics of Rad51 filament formation on ssDNA and disruption by Srs2. Rad51 forms filaments on ssDNA by sequential binding of primarily monomers and dimers in a 5’ to 3’ direction. One Rad51 molecule binds 3 nucleotides and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent ssDNA translocation and repetitive scrunching and this latter activity is exploited to prevent Rad51 filament reformation. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2's DNA unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates Rad51 filament formation.
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