Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo
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ABSTRACT:
SUBMITTER: Rebecca C. Burgess
PROVIDER: S-JCBD-200810055 | BioStudies | 2009-06-08
REPOSITORIES: biostudies
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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 srs2? phenotypes in haploid cells, such as UV and ?-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 srs2? 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 srs2? 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 srs2? 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.
2013-01-01 | S-EPMC3794917 | BioStudies
Project description:The Shu complex, which contains RAD51 paralogues, is involved in the decision between homologous recombination and error-prone repair. We discovered a link to ribosomal DNA (rDNA) recombination when we found an interaction between one member of the Shu complex, SHU1, and UAF30, a component of the upstream activating factor complex (UAF), which regulates rDNA transcription. In the absence of Uaf30, rDNA copy number increases, and this increase depends on several functional subunits of the Shu complex. Furthermore, in the absence of Uaf30, we find that Shu1 and Srs2, an anti-recombinase DNA helicase with which the Shu complex physically interacts, act in the same pathway regulating rDNA recombination. In addition, Shu1 modulates Srs2 recruitment to both induced and spontaneous foci correlating with a decrease in Rad51 foci, demonstrating that the Shu complex is an important regulator of Srs2 activity. Last, we show that Shu1 regulation of Srs2 to double-strand breaks is not restricted to the rDNA, indicating a more general function for the Shu complex in the regulation of Srs2. We propose that the Shu complex shifts the balance of repair toward Rad51 filament stabilization by inhibiting the disassembly reaction of Srs2.
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Project description:Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In <i>Saccharomyces cerevisiae</i>, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.
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Project description:A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.
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Project description:Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2?), and diploid srs2? mus81? mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81-Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability.
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Project description:DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNA-mediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.
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Project description:Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv.
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