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.     

Interaction between yeast sgs1 helicase and DNA topoisomerase III

The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins. In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo. Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III. Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA. A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays. Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues. In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant. These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant.     

Sgs1, a Homologue of the Bloom''s and Werner''s Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.     

The absence of Top3 reveals an interaction between the Sgs1 and Pif1 DNA helicases in Saccharomyces cerevisiae

RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pif1, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1-Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.     

Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.

Topoisomerases catalyse changes in the topological state of DNA and are required for many aspects of DNA metabolism. While the functions of topoisomerases I and II in eukaryotes are well established, the role of topoisomerase III remains poorly defined. We have identified a gene in the fission yeast Schizosaccharomyces pombe, designated top3 (+), which shows significant sequence similarity to genes encoding topoisomerase III enzymes in other eukaryotic species. In common with murine TOP3 alpha, but in contrast to Saccharomyces cerevisiae TOP3, the S.pombe top3 (+)gene is essential for long-term cell viability. Fission yeast haploid spores containing a disrupted top3 (+)gene germinate successfully, but then undergo only a limited number of cell divisions. Analysis of these top3 mutants revealed evidence of aberrant mitotic chromosome segregation, including the 'cut' phenotype, where septation is completed prior to nuclear division. Consistent with the existence of an intimate association (originally identified in S.cerevisiae ) between topoisomerase III and DNA helicases of the RecQ family, deletion of the rqh1 (+)gene encoding the only known RecQ helicase in S.pombe suppresses lethality in top3 mutants. This conservation of genetic interaction between two widely diverged yeasts suggests that the RecQ family helicases encoded by the Bloom's and Werner's syndrome genes are likely to act in concert with topoisomerase III isozymes in human cells. Our data are consistent with a model in which the association of a RecQ helicase and topoisomerase III is important for facilitating decatenation of late stage replicons to permit faithful chromosome segregation during anaphase.     

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.     

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.     

Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants

The Saccharomyces cerevisiae TOP3 gene encodes the type IA topoisomerase (Top3p) that is highly conserved in evolution. Deletion of TOP3 leads to a reduction in cell viability, hyper-recombination between repetitive DNA sequences, and abnormalities in both cell cycle progression and responses to DNA damaging agents. Deletion of SGS1, encoding the sole RecQ family helicase in S. cerevisiae, strongly suppresses the phenotypic effects of loss of TOP3 function. Here, we show that many of the adverse phenotypic effects of TOP3 deletion can also be partially alleviated by disruption of homologous recombination (HR) functions. This genetic interaction is seen both in strains deleted for TOP3 and in wild-type strains over-expressing a dominant-negative Top3p mutant form that confers a top3-like phenotype. Moreover, we show that this genetic interaction is conserved in the distantly-related fission yeast, Schizosaccharomyces pombe. Our results implicate topoisomerase III enzymes in recombination repair events required for cellular protection against DNA damaging agents and DNA replication inhibitors.     

The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.

We have previously shown that cells mutant for TOP3, a gene encoding a prokaryotic-like type I topoisomerase in Saccharomyces cerevisiae, display a pleiotropic phenotype including slow growth and genome instability. We identified a mutation, sgs1 (slow growth suppressor), that suppresses both the growth defect and the increased genomic instability of top3 mutants. Here we report the independent isolation of the SGS1 gene in a screen for proteins that interact with Top3. DNA sequence analysis reveals that the putative Sgs1 protein is highly homologous to the helicase encoded by the Escherichia coli recQ gene. These results imply that Sgs1 creates a deleterious topological substrate that Top3 preferentially resolves. The interaction of the Sgs1 helicase homolog and the Top3 topoisomerase is reminiscent of the recently described structure of reverse gyrase from Sulfolobus acidocaldarius, in which a type I DNA topoisomerase and a helicase-like domain are fused in a single polypeptide.     

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.     

Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair,somatic and meiotic recombination

Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.     

The RecQ gene family in plants

RecQ helicases are conserved throughout all kingdoms of life regarding their overall structure and function. They are 3'-5' DNA helicases resolving different recombinogenic DNA structures. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In eukaryotes the number of RecQ genes and the structure of RecQ proteins vary strongly between organisms. Therefore, they have been named RecQ-like genes. Knockouts of several RecQ-like genes cause severe diseases in animals or harmful cellular phenotypes in yeast. Until now the largest number of RecQ-like genes per organism has been found in plants. Arabidopsis and rice possess seven different RecQ-like genes each. In the almost completely sequenced genome of the moss Physcomitrella patens at least five RecQ-like genes are present. One of the major present and future research aims is to define putative plant-specific functions and to assign their roles in DNA repair and recombination pathways in relation to RecQ genes from other eukaryotes. Regarding their intron positions, the structures of six RecQ-like genes of dicots and monocots are virtually identical indicating a conservation over a time scale of 150 million years. In contrast to other eukaryotes one gene (RecQsim) exists exclusively in plants. It possesses an interrupted helicase domain but nevertheless seems to have maintained the RecQ function. Owing to a recent gene duplication besides the AtRecQl4A gene an additional RecQ-like gene (AtRecQl4B) exists in the Brassicaceae only. Genetic studies indicate that a AtRecQl4A knockout results in sensitivity to mutagens as well as an hyper-recombination phenotype. Since AtRecQl4B was still present, both genes must have non-redundant roles. Analysis of plant RecQ-like genes will not only increase the knowledge on DNA repair and recombination, but also on the evolution and radiation of protein families.     

A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.     

Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity

RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3alpha, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Delta. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a "random walk," and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Delta. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.     

Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.     

Growth defect and mutator phenotypes of RecQ-deficient Neurospora crassa mutants separately result from homologous recombination and nonhomologous end joining during repair of DNA double-strand breaks

RecQ helicases function in the maintenance of genome stability in many organisms. The filamentous fungus Neurospora crassa has two RecQ homologs, QDE3 and RECQ2. We found that the qde-3 recQ2 double mutant showed a severe growth defect. The growth defect was alleviated by mutation in mei-3, the homolog of yeast RAD51, which is required for homologous recombination (HR), suggesting that HR is responsible for this phenotype. We also found that the qde-3 recQ2 double mutant showed a mutator phenotype, yielding mostly deletions. This phenotype was completely suppressed by mutation of mus-52, a homolog of the human KU80 gene that is required for nonhomologous end joining (NHEJ), but was unaffected by mutation of mei-3. The high spontaneous mutation frequency in the double mutant is thus likely to be due to NHEJ acting on an elevated frequency of double-strand breaks (DSBs) and we therefore suggest that QDE3 and RECQ2 maintain chromosome stability by suppressing the formation of spontaneous DSBs.     

RecQ helicases in DNA double strand break repair and telomere maintenance

Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.