The yeast Sgs1 helicase is differentially required for genomic and ribosomal DNA replication

The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1Delta cells. Unexpectedly, we find that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1Delta cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage

In the budding yeast Saccharomyces cerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage. A second helicase, Sgs1, is the S.cerevisiae homologue of the human BLM and WRN proteins, which are defective in cancer predisposition and/or premature ageing syndromes. Saccharomyces cerevisiae cells lacking both Srs2 and Sgs1 exhibit a severe growth defect. We have identified an Srs2 orthologue in the fission yeast Schizosaccharomyces pombe, and have investigated its role in responses to UV irradiation and inhibition of DNA replication. Deletion of fission yeast srs2 caused spontaneous hyper-recombination and UV sensitivity, and simultaneous deletion of the SGS1 homologue rqh1 caused a severe growth defect reminiscent of that seen in the equivalent S.cerevisiae mutant. However, unlike in budding yeast, inactivation of the homologous recombination pathway did not suppress this growth defect. Indeed, the homologous recombination pathway was required for maintenance of normal fission yeast viability in the absence of Srs2, and loss of homologous recombination and loss of Srs2 contributed additively to UV sensitivity. We conclude that Srs2 plays related, but not identical, roles in the two yeast species.     

The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund-Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele-replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NDelta) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Delta mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein-protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.     

rqh1+, a fission yeast gene related to the Bloom''s and Werner''s syndrome genes, is required for reversible S phase arrest.

In eukaryotic cells, S phase can be reversibly arrested by drugs that inhibit DNA synthesis or DNA damage. Here we show that recovery from such treatments is under genetic control and is defective in fission yeast rqh1 mutants. rqh1+, previously known as hus2+, encodes a putative DNA helicase related to the Escherichia coli RecQ helicase, with particular homology to the gene products of the human BLM and WRN genes and the Saccharomyces cerevisiae SGS1 gene. BLM and WRN are mutated in patients with Bloom's syndrome and Werner's syndrome respectively. Both syndromes are associated with genomic instability and cancer susceptibility. We show that, like BLM and SGS1, rqh1+ is required to prevent recombination and that in fission yeast suppression of inappropriate recombination is essential for reversible S phase arrest.     

RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex

SGS1 encodes a DNA helicase whose homologues in human cells include the BLM, WRN, and RECQ4 genes, mutations in which lead to cancer-predisposition syndromes. Clustering of synthetic genetic interactions identified by large-scale genetic network analysis revealed that the genetic interaction profile of the gene RMI1 (RecQ-mediated genome instability, also known as NCE4 and YPL024W) was highly similar to that of SGS1 and TOP3, suggesting a functional relationship between Rmi1 and the Sgs1/Top3 complex. We show that Rmi1 physically interacts with Sgs1 and Top3 and is a third member of this complex. Cells lacking RMI1 activate the Rad53 checkpoint kinase, undergo a mitotic delay, and display increased relocalization of the recombination repair protein Rad52, indicating the presence of spontaneous DNA damage. Consistent with a role for RMI1 in maintaining genome integrity, rmi1Delta cells exhibit increased recombination frequency and increased frequency of gross chromosomal rearrangements. In addition, rmi1Delta strains fail to fully activate Rad53 upon exposure to DNA-damaging agents, suggesting that Rmi1 is also an important part of the Rad53-dependent DNA damage response.     

Examination of the roles of Sgs1 and Srs2 helicases in the enforcement of recombination fidelity in Saccharomyces cerevisiae

Mutation in SGS1, which encodes the yeast homolog of the human Bloom helicase, or in mismatch repair (MMR) genes confers defects in the suppression of mitotic recombination between similar but nonidentical (homeologous) sequences. Mutational analysis of SGS1 suggests that the helicase activity is required for the suppression of both homologous and homeologous recombination and that the C-terminal 200 amino acids may be required specifically for the suppression of homeologous recombination. To clarify the mechanism by which the Sgs1 helicase enforces the fidelity of recombination, we examined the phenotypes associated with SGS1 deletion in MMR-defective and recombination-defective backgrounds. Deletion of SGS1 caused no additional loss of recombination fidelity above that associated with MMR defects, indicating that the suppression of homeologous recombination by Sgs1 may be dependent on MMR. However, the phenotype of the sgs1 rad51 mutant suggests a MMR-independent role of Sgs1 in the suppression of RAD51-independent recombination. While homologous recombination levels increase in sgs1Delta and in srs2Delta strains, the suppression of homeologous recombination was not relaxed in the srs2 mutant. Thus, although both Sgs1 and Srs2 limit the overall level of mitotic recombination, there are distinct differences in the roles of these helicases with respect to enforcement of recombination fidelity.     

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.     

Regulation of alternative replication bypass pathways at stalled replication forks and its effects on genome stability: a yeast model

Replication-blocking lesions result in increased genomic instability by stalling replication forks. Eukaryotic cells appear to have evolved several surveillance and repair/bypass mechanisms to ensure that replication can be resumed at these stalled forks. In the yeast Saccharomyces cerevisiae, the helicases Srs2 and Sgs1 appear to play a role in controlling the processing and stabilization of stalled replication forks. These proteins appear to be tightly regulated throughout the cell cycle and play a direct role in DNA-damage checkpoints. This allows the cells to determine the best mechanism to reestablish replication at the stalled fork: by shuttling the lesion into the RAD6-dependent pathway that can lead to error-free or error-prone bypass; or by using homologous recombination. Under conditions where both the RAD6-dependent pathway and recombination are disabled, the cells can bypass the lesion using a novel damage avoidance mechanism that is controlled by Mgs1. Replication fork bypass processes appear to be highly conserved within eukaryotes, with homologs for SGS1 and MGS1 found in both Schizosaccharomyces pombe and mammalian cells.     

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.     

Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae

In budding yeast, loss of topoisomerase III, encoded by the TOP3 gene, leads to a genomic instability phenotype that includes slow growth, hyper-sensitivity to genotoxic agents, mitotic hyper-recombination, increased chromosome missegregation, and meiotic failure. Slow growth and other defects of top3 mutants are suppressed by mutation of SGS1, which encodes the only RecQ helicase in S. cerevisiae. sgs1 is epistatic to top3, suggesting that the two proteins act in the same pathway. To identify other factors that function in the Sgs1-Top3 pathway, we undertook a genetic screen for non-sgs1 suppressors of top3 defects. We found that slow growth and DNA damage sensitivity of top3 mutants are suppressed by mutations in RAD51, RAD54, RAD55, and RAD57. In contrast, top3 mutants show extreme synergistic growth defects with mutations in RAD50, MRE11, XRS2, RDH54, and RAD1. We also analyzed recombination at the SUP4-o region, showing that in a rad51, rad54, rad55, or rad57 background top3Delta does not increase recombination to the same degree as in a wild-type strain. These results suggest that the presence of the Rad51 homologous recombination complex in a top3 background facilitates creation of detrimental intermediates by Sgs1. We present a model wherein Rad51 helps recruit Sgs1-Top3 to sites of replicative damage.     

Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex

Genome stability requires a set of RecQ-Top3 DNA helicase-topoisomerase complexes whose sole budding yeast homolog is encoded by SGS1-TOP3. RMI1/NCE4 was identified as a potential intermediate in the SGS1-TOP3 pathway, based on the observation that strains lacking any one of these genes require MUS81 and MMS4 for viability. This idea was tested by confirming that sgs1 and rmi1 mutants display the same spectrum of synthetic lethal interactions, including the requirements for SLX1, SLX4, SLX5, and SLX8, and by demonstrating that rmi1 mus81 synthetic lethality is dependent on homologous recombination. On their own, mutations in RMI1 result in phenotypes that mimic those of sgs1 or top3 strains including slow growth, hyperrecombination, DNA damage sensitivity, and reduced sporulation. And like top3 strains, most rmi1 phenotypes are suppressed by mutations in SGS1. We show that Rmi1 forms a heteromeric complex with Sgs1-Top3 in yeast and that these proteins interact directly in a recombinant system. The Rmi1-Top3 complex is stable in the absence of the Sgs1 helicase, but the loss of either Rmi1 or Top3 in yeast compromises its partner's interaction with Sgs1. Biochemical studies demonstrate that recombinant Rmi1 is a structure-specific DNA binding protein with a preference for cruciform structures. We propose that the DNA binding specificity of Rmi1 plays a role in targeting Sgs1-Top3 to appropriate substrates.     

The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs

The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase.     

Sumoylation of the BLM ortholog,Sgs1, promotes telomere–telomere recombination in budding yeast

BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to be uniquely required for Sgs1’s role in telomere–telomere recombination. In contrast, sumoylation was dispensable for Sgs1’s roles in DNA damage tolerance, supppression of direct repeat and rDNA recombination, and promotion of top3Δ slow growth. Our results demonstrate that although modification by SUMO is a conserved feature of RecQ family DNA helicases, the major sites of modification are located on different domains of the protein in different organisms. We suggest that sumoylation of different domains of RecQ DNA helicases from different organisms contributes to conserved roles in regulating telomeric recombination.     

A novel protein interacts with the Werner's syndrome gene product physically and functionally

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.     

Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.     

Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1

The Saccharomyces cerevisiae gene SGS1 encodes a DNA helicase that shows homology to the Escherichia coli protein RecQ and the products of the BLM and WRN genes in humans, which are defective in Bloom's and Werner's syndrome, respectively. Recently, it has been proposed that this helicase is involved in maintaining the integrity of the rDNA and that loss of Sgs1 function leads to accelerated aging. Sgs1 has been isolated on the basis of its genetic interaction with both topoisomerase I and topoisomerase III, as well as in a two-hybrid screen for proteins that interact with the C-terminal portion of topoisomerase II. We have defined the minimal structural elements of Sgs1 required for its interactions with the three topoisomerases, and demonstrate that the complex phenotypes associated with sgs1 mutants are a consequence of a dysfunctional Sgs1-Top3 complex. We also report that the synthetic relationship between mutations in SGS1 and SRS2, which encodes another helicase implicated in recombinational repair, likewise result from a dysfunctional Sgs1-Top3 interaction. Our findings indicate that Sgs1 may act on different DNA structures depending on the activity of topoisomerase I, Srs2 and topoisomerase III.     

The ability of Sgs1 to interact with DNA topoisomerase III is essential for damage-induced recombination

SGS1 encodes a protein having DNA helicase activity, and a mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. In this study, we examined whether Sgs1 prevents formation of DNA double strand breaks (DSBs) or is involved in DSB repair following exposure to methyl methanesulfonate (MMS). An analysis by pulsed-field gel electrophoresis and epistasis analyses indicated that Sgs1 is required for DSB repair that involves Rad52. In addition, analyses on the relationship between Sgs1 and proteins involved in DSB repair suggested that Sgs1 and Mre11 function via independent pathways both of which require Rad52. In sgs1 mutants, interchromosomal heteroallelic recombination and sister chromatid recombination (SCR) were not induced upon exposure to MMS, though both were induced in wild type cells, indicating the involvement of Sgs1 in heteroallelic recombination and SCR. Surprisingly, the ability of Sgs1 to bind to DNA topoisomerase III (Top3) was absolutely required for the induction of heteroallelic recombination and SCR and suppression of MMS sensitivity but its helicase activity was not, suggesting that Top3 plays a more important role in both recombinations than the DNA helicase activity of Sgs1.     

Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair

Mutations in human homologues of the bacterial RecQ helicase cause diseases leading to cancer predisposition and/or shortened lifespan (Werner, Bloom, and Rothmund–Thomson syndromes). The budding yeast Saccharomyces cerevisiae has one RecQ helicase, Sgs1, which functions with Top3 and Rmi1 in DNA repair. Here, we report separation‐of‐function alleles of SGS1 that suppress the slow growth of top3Δ and rmi1Δ cells similar to an SGS1 deletion, but are resistant to DNA damage similar to wild‐type SGS1. In one allele, the second acidic region is deleted, and in the other, only a single aspartic acid residue 664 is deleted. sgs1‐D664Δ, unlike sgs1Δ, neither disrupts DNA recombination nor has synthetic growth defects when combined with DNA repair mutants. However, during S phase, it accumulates replication‐associated X‐shaped structures at damaged replication forks. Furthermore, fluorescent microscopy reveals that the sgs1‐D664Δ allele exhibits increased spontaneous RPA foci, suggesting that the persistent X‐structures may contain single‐stranded DNA. Taken together, these results suggest that the Sgs1 function in repair of DNA replication intermediates can be uncoupled from its role in homologous recombinational repair.     

Werner complex deficiency in cells disrupts the Nuclear Pore Complex and the distribution of lamin B1

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of “RAN holes” void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus.