Srs2 Helicase Research Articles

WRN Interacts Physically and Functionally with the Recombination Mediator Protein RAD52

Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.

The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments.

Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures. Here we show that DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and that Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control.

Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae.

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5' and 3', are rapidly degraded. This is not due to the inability to recombine, since the 3' single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.

Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication.

Toxic recombination events are detected in vegetative Saccharomyces cerevisiae cells through negative growth interactions between certain combinations of mutations. For example, mutations affecting both the Srs2 and Sgs1 helicases result in extremely poor growth, a phenotype suppressed by mutations in genes that govern early stages of recombination. Here, we identify a similar interaction involving double mutations affecting Sgs1 or Top3 and Mus81 or Mms4. We also find that the primary DNA structures that initiate these toxic recombination events cannot be double-strand breaks and thus are likely to be single-stranded DNA. We interpret our results in the context of the idea that replication stalling leaves single-stranded DNA, which can then be processed by two competing mechanisms: recombination and nonrecombination gap-filling. Functions involved in preventing toxic recombination would either avoid replicative defects or act on recombination intermediates. Our results suggest that Srs2 channels recombination intermediates back into the gap-filling route, whereas Sgs1Top3 and Mus81Mms4 are involved in recombination andor in replication to allow replication restart.

Recovery from Checkpoint-Mediated Arrest after Repair of a Double-Strand Break Requires Srs2 Helicase

In Saccharomyces strains in which homologous recombination is delayed sufficiently to activate the DNA damage checkpoint, Rad53p checkpoint kinase activity appears 1 hr after DSB induction and disappears soon after completion of repair. Cells lacking Srs2p helicase fail to recover even though they apparently complete DNA repair; Rad53p kinase remains activated. srs2Δ cells also fail to adapt when DSB repair is prevented. The recovery defect of srs2Δ is suppressed in mec1Δ strains lacking the checkpoint or when DSB repair occurs before checkpoint activation. Permanent preanaphase arrest of srs2Δ cells is reversed by the addition of caffeine after cells have arrested. Thus, in addition to its roles in recombination, Srs2p appears to be needed to turn off the DNA damage checkpoint.

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.

Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases.

DNA helicases are involved in many aspects of DNA metabolism, including transcription, replication, recombination and repair. In the yeast Saccharomyces cerevisiae, the absence of the Sgs1 helicase results in genomic instability and accelerated ageing. In human cells, mutations in orthologues of SGS1 lead to Bloom (BS), Werner (WS) or Rothmund-Thomson (RTS) syndromes, which are rare, autosomal recessive diseases characterized by genetic instability associated with cancer predisposition. Although data concerning these human diseases are accumulating, there is still no clear idea of the function of the proteins involved. Here we show that sgs1Delta mutants are deficient in DNA repair and are defective for induced recombination events that involve homologous chromosomes. The role of homologous recombination is further evidenced in haploid cells in which both Sgs1p and Srs2p are absent. Yeast SRS2 encodes another DNA helicase involved in the maintenance of genome integrity. Our data suggest that some defects observed in BS, WS or RTS are the consequence of unrestrained recombination.

Genetic interactions between mutants of the `error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis

We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation [B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199–1211.]. In the present study we characterize the effect of mutations in genes of the `error-prone' or postreplicative repair group on recombination and mutation. We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats. The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination. In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen. The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase. We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes. The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes. The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products.

Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase.

Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad5l, a homolog of the bacterial RecA protein. The nature of these mutations was determined by direct sequencing. The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites. The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis. Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions. By themselves, all the alleles confer a rad5l null phenotype. When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes. This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions.

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation.

The expression of the SRS2 gene, which encodes a DNA helicase involved in DNA repair in Saccharomyces cerevisiae, was studied using an SRS2-lacZ fusion integrated at the chromosomal SRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression of SRS2 rises at a time nearly coincident with commitment to recombination. Since srs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity of G1 cells or Srs2 functions in a repair mechanism associated with replication.

Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins.

Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.

Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins.

Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.