Dataset Information

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

ABSTRACT: Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.

SUBMITTER: Meir A

PROVIDER: S-EPMC8795549 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Publications

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Meir Aviv A Crickard J Brooks JB Kwon Youngho Y Sung Patrick P Greene Eric C EC

Proceedings of the National Academy of Sciences of the United States of America 20220101 4

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombin ...[more]

PMID: 35042797

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Project description:The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.

Dataset Information

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Publications

Rad54 and Rdh54 prevent Srs2-mediated disruption of Rad51 presynaptic filaments.

Similar Datasets

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