Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In Schizosaccharomyces pombe, topoisomerase III is encoded by a single gene, top3(+), which is essential for cell viability and proper chromosome segregation. Deletion of rqh1(+), which encodes the sole RecQ family helicase in S. pombe, suppresses the lethality caused by loss of top3. Here, we provide evidence suggesting that the lethality in top3 mutants is due to accumulation of aberrant DNA structures that arise during S phase, as judged by pulsed-field gel electrophoresis. Using a top3 shut-off strain, we show here that depletion of Top3 activates the DNA damage checkpoint associated with phosphorylation of the checkpoint kinase Chk1. Despite activation of this checkpoint, top3 cells exit the arrest but fail to undergo faithful chromosome segregation. However, these mitotic defects are secondary to chromosomal abnormalities that lead to the lethality, because advance into mitosis did not adversely affect cell survival. Furthermore, top3 function is required for maintenance of nucleolar structure, possibly due to its ability to prevent recombination at the rDNA loci. Our data are consistent with the notion that Top3 has a key function in homologous recombinational repair during S phase that is essential for ensuring subsequent fidelity of chromosome segregation.
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PMID:Requirement for Schizosaccharomyces pombe Top3 in the maintenance of chromosome integrity. 1534 8

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.
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PMID:Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. 1561 82

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.
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PMID:Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination. 1564 19

Helicases of the RecQ family and topoisomerase III are evolutionarily conserved proteins important for maintenance of genome stability. In Saccharomyces cerevisiae, loss of the TOP3 gene, encoding topoisomerase III, results in a phenotype of slow growth, DNA damage sensitivity, meiotic defects, and hyperrecombination. The sole RecQ helicase in budding yeast, Sgs1, interacts with Top3 both physically and genetically, and the two proteins are thought to act in concert in vivo. Much recent genetic and biochemical evidence points to the role of RecQ helicases and topoisomerase III in regulating homologous recombination (HR) during DNA replication. Previously, we found that mutations in HR genes partially suppress top3 slow growth. Here, we describe the analysis of four additional mutational suppressors of top3 defects: shu1, shu2, psy3, and csm2. These genes belong to one epistasis group and their protein products interact with each other, strongly suggesting that they function as a complex in vivo. Their mutant phenotype indicates that they are important for error-free repair of spontaneous and induced DNA lesions, protecting the genome from mutation. These mutants exhibit an epistatic relationship with rad52 and show altered dynamics of Rad52-YFP foci, suggesting a role for these proteins in recombinational repair.
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PMID:A genetic screen for top3 suppressors in Saccharomyces cerevisiae identifies SHU1, SHU2, PSY3 and CSM2: four genes involved in error-free DNA repair. 1565 96

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.
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PMID:Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus. 1567 17

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.
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PMID:Yeast Rmi1/Nce4 controls genome stability as a subunit of the Sgs1-Top3 complex. 1589 53

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution. Here, we show that dissolution is highly specific for BLM among human RecQ helicases and critically depends upon a functional HRDC domain in BLM. We show that the HRDC domain confers DNA structure specificity, and is required for the efficient binding to and unwinding of double Holliday junctions, but not for the unwinding of a simple partial duplex substrate. Furthermore, we show that lysine-1270 of BLM, which resides in the HRDC domain and is predicted to play a role in mediating interactions with DNA, is required for efficient dissolution.
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PMID:The HRDC domain of BLM is required for the dissolution of double Holliday junctions. 1599 Aug 71

The RecQ family of DNA helicases is highly conserved in evolution from bacteria to humans. Of the five known human RecQ family members, three (BLM, WRN and RECQ4, which cause Bloom's syndrome, Werner's syndrome and Rothmund-Thomson syndrome respectively) are mutated in distinct clinical disorders associated with cancer predisposition and/or premature aging. BLM forms part of a multienzyme complex including topoisomerase IIIalpha, replication protein A and a newly identified factor called BLAP75. Together, these proteins play a role in the resolution of DNA structures that arise during the process of homologous recombination repair. In the absence of BLM, cells show genomic instability and a high incidence of sister-chromatid exchanges. In addition to a DNA structure-specific helicase activity, BLM also catalyses Holliday-junction branch migration and the annealing of complementary single-stranded DNA molecules.
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PMID:Roles of the Bloom's syndrome helicase in the maintenance of genome stability. 1624 45

RecQ DNA helicases, including yeast Sgs1p and the human Werner and Bloom syndrome proteins, participate in telomere biology, but the underlying mechanisms are not fully understood. Here, we explore the protein sequences and genetic interactors of Sgs1p that function to slow the senescence of telomerase (tlc1) mutants. We find that the S-phase checkpoint function of Sgs1p is dispensable for preventing rapid senescence, but that Sgs1p sequences required for homologous recombination, including the helicase domain and topoisomerase III interaction domain, are essential. sgs1 and rad52 mutations are epistatic during senescence, indicating that Sgs1p participates in a RAD52-dependent recombinational pathway of telomere maintenance. Several mutations that are synthetically lethal with sgs1 mutation and which individually lead to genome instability, including mus81, srs2, rrm3, slx1 and top1, do not speed the senescence of tlc1 mutants, indicating that the rapid senescence of sgs1 tlc1 mutants is not caused by generic genome instability. However, mutations in SLX5 or SLX8, which encode proteins that function together in a complex that is required for viability in sgs1 mutants, do speed the senescence of tlc1 mutants. These observations further define roles for RecQ helicases and related proteins in telomere maintenance.
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PMID:Evidence that the S.cerevisiae Sgs1 protein facilitates recombinational repair of telomeres during senescence. 1642 46

BLM encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of BLM gives rise to the cancer predisposition disorder Bloom's syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom's syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase IIIalpha (hTOPO IIIalpha), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM/hTOPO IIIalpha complex, BLAP75/RMI1, promotes dissolution catalyzed by hTOPO IIIalpha. This activity of BLAP75/RMI1 is specific for dissolution catalyzed by hTOPO IIIalpha because it has no effect in reactions containing either Escherichia coli Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75/RMI1 acts by recruiting hTOPO IIIalpha to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75/RMI1 might confer specificity on the execution of this process are discussed.
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PMID:BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates. 1653 86


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