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Remodeling of the Rad51 DNA strand-exchange protein by the Srs2 helicase.

ABSTRACT: Homologous recombination is associated with the dynamic assembly and disassembly of DNA-protein complexes. Assembly of a nucleoprotein filament comprising ssDNA and the RecA homolog, Rad51, is a key step required for homology search during recombination. The budding yeast Srs2 DNA translocase is known to dismantle Rad51 filament in vitro. However, there is limited evidence to support the dismantling activity of Srs2 in vivo. Here, we show that Srs2 indeed disrupts Rad51-containing complexes from chromosomes during meiosis. Overexpression of Srs2 during the meiotic prophase impairs meiotic recombination and removes Rad51 from meiotic chromosomes. This dismantling activity is specific for Rad51, as Srs2 Overexpression does not remove Dmc1 (a meiosis-specific Rad51 homolog), Rad52 (a Rad51 mediator), or replication protein A (RPA; a single-stranded DNA-binding protein). Rather, RPA replaces Rad51 under these conditions. A mutant Srs2 lacking helicase activity cannot remove Rad51 from meiotic chromosomes. Interestingly, the Rad51-binding domain of Srs2, which is critical for Rad51-dismantling activity in vitro, is not essential for this activity in vivo. Our results suggest that a precise level of Srs2, in the form of the Srs2 translocase, is required to appropriately regulate the Rad51 nucleoprotein filament dynamics during meiosis.

SUBMITTER: Sasanuma H

PROVIDER: S-EPMC3730916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5'-3' direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. 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 the Rad51 filament formation.

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