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)

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative (ALT) recombination mechanism. In yeast, Sgs1p and its associated type IA topoisomerase, Top3p, may work coordinately in removing Holliday junction intermediates from a crossover-producing recombination pathway. Previous studies have also indicated that Sgs1 helicase acts in a telomere recombination pathway. Here we show that topoisomerase III is involved in telomere-telomere recombination. The recovery of telomere recombination-dependent survivors in a telomerase-minus yeast strain was dependent on Top3p catalytic activity. Moreover, the RIF1 and RIF2 genes are required for the establishment of TOP3/SGS1-dependent telomere-telomere recombination. In human Saos-2 ALT cells, human topoisomerase IIIalpha (hTOP3alpha) also contributes to telomere recombination. Strikingly, the telomerase activity is clearly enhanced in surviving si-hTOP3alpha Saos-2 ALT cells. Altogether, the present results suggest a potential role for hTOP3alpha in dissociating telomeric structures in telomerase-deficient cells, providing therapeutic implications in human tumors.
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PMID:Involvement of topoisomerase III in telomere-telomere recombination. 1654 98

Bloom syndrome (BS), an autosomal recessive disorder, is marked by a high incidence of cancer early in life. Cells derived from BS patients are unstable genetically and exhibit frequent sister chromatid exchanges, reflective of homologous recombination (HR) deregulation. BLM, the RecQ-like helicase mutated in BS, is found in several cellular protein complexes, all of which contain topoisomerase IIIalpha (Topo IIIalpha) and a novel protein BLAP75. Here, using highly purified human proteins, we show that BLAP75 associates independently with both Topo IIIalpha and BLM. Even though BLM and Topo IIIalpha can dissolve the double Holliday junction (DHJ) to yield non-crossover recombinants (1), under physiological conditions, DHJ dissolution becomes completely dependent on BLAP75. The effect of BLAP75 on BLM-Topo IIIalpha is highly specific, as it is not seen with the combination of Topo IIIalpha and Escherichia coli RecQ helicase or another human RecQ-like helicase WRN. Thus, BLM, Topo IIIalpha, and BLAP75 constitute a dissolvasome complex that processes HR intermediates to limit DNA crossover formation. This function of the BLM-Topo IIIalpha-BLAP75 dissolvasome is likely indispensable for genome maintenance and cancer avoidance.
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PMID:A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIalpha, and BLAP75. 1659 95

The Bloom's syndrome helicase, BLM, is a member of the highly conserved RecQ family, and possesses both DNA unwinding and DNA strand annealing activities. BLM also promotes branch migration of Holliday junctions. One role for BLM is to act in conjunction with topoisomerase IIIalpha to process homologous recombination (HR) intermediates containing a double Holliday junction by a process termed dissolution. However, several lines of evidence suggest that BLM may also act early in one or more of the recombination pathways to eliminate illegitimate or aberrantly paired DNA joint molecules. We have investigated whether BLM can disrupt DNA displacement loops (D-loops), which represent the initial strand invasion step of HR. We show that mobile D-loops created by the RecA recombinase are a highly preferred substrate for BLM with the invading strand being displaced from the duplex. We have identified structural features of the D-loop that determine the efficiency with which BLM promotes D-loop dissociation. We discuss these results in the context of models for the role of BLM as an 'anti-recombinase'.
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PMID:Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. 1667 Apr 33

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.
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PMID:Sgs1 regulates gene conversion tract lengths and crossovers independently of its helicase activity. 1670 62

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A "left"-"right" bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47(-)39(-), uvsX 39(-) and 59(-)39(-) are lethal.
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PMID:Double-strand break repair in bacteriophage T4: coordination of DNA ends and effects of mutations in recombinational genes. 1671 67

Werner syndrome (WS) is an inherited disorder characterized by premature onset of aging, genomic instability, and increased cancer incidence. The disease is caused by loss of function mutations of the WRN gene, a RecQ family member with both helicase and exonuclease activities. However, despite its putative tumor-suppressor function, little is known about the contribution of WRN to human sporadic malignancies. Here, we report that WRN function is abrogated in human cancer cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of WRN leads to the loss of WRN-associated exonuclease activity and increased chromosomal instability and apoptosis induced by topoisomerase inhibitors. The described phenotype is reversed by the use of a DNA-demethylating agent or by the reintroduction of WRN into cancer cells displaying methylation-dependent silencing of WRN. Furthermore, the restoration of WRN expression induces tumor-suppressor-like features, such as reduced colony formation density and inhibition of tumor growth in nude mouse xenograft models. Screening a large collection of human primary tumors (n = 630) from different cell types revealed that WRN CpG island hypermethylation was a common event in epithelial and mesenchymal tumorigenesis. Most importantly, WRN hypermethylation in colorectal tumors was a predictor of good clinical response to the camptothecin analogue irinotecan, a topoisomerase inhibitor commonly used in the clinical setting for the treatment of this tumor type. These findings highlight the importance of WRN epigenetic inactivation in human cancer, leading to enhanced chromosomal instability and hypersensitivity to chemotherapeutic drugs.
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PMID:Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer. 1672 99

It has long been suspected that a double Holliday junction (dHJ) could be resolved by a topoisomerase partnered with a helicase by convergent branch migration of the HJs. Genetic analysis of yeast TOP3 and SGS1 has lent considerable evidence to the notion that the protein products of these genes are involved in just such a process, although biochemical analysis of the metabolism of a dHJ has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a dHJ substrate that recapitulates many of the features of an endogenous dHJ and represents a much earlier intermediate in the resolution pathway. Here, we show that Drosophila topoisomerase IIIalpha (Topo IIIalpha) and Blm (a homolog of Sgs1) are capable of resolving this substrate to non-cross-over products and that this activity is stimulated by replication protein A (RPA). We investigated the ability of other Drosophila topoisomerases to perform this reaction in concert with Blm and RPA and discovered that this resolution activity is unique to Topo IIIalpha. Examination of the mechanism of resolution reveals that Topo IIIalpha, Blm, and RPA resolve this substrate by convergent migration of the two HJs toward each other, collapsing the dHJ. This mechanism stands in contrast to classic resolvase activities that use a structure-specific endonuclease to cleave the HJs.
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PMID:Topoisomerase IIIalpha and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration. 1684 22

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.
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PMID:Bloom helicase and DNA topoisomerase IIIalpha are involved in the dissolution of sister chromatids. 1688 May 37

RecG is a member of the superfamily 2 helicase family. Its possible role in vivo is ATP hydrolysis driven regression of stalled replication forks. To gain mechanistic insight into how this is achieved, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of RecG in vitro. The results demonstrate an overwhelming preference for negatively supercoiled DNA ((-)scDNA) as a cofactor for the hydrolysis of ATP. In the presence of (-)scDNA the catalytic efficiency of RecG and the processivity (as revealed through heparin trapping), were higher than on any other cofactor examined. The activity of RecG on (-)scDNA was not due to the presence of single-stranded regions functioning as loading sites for the enzyme as relaxed circular DNA treated with DNA gyrase, resulted in the highest levels of ATPase activity. Relaxation of (-)scDNA by a topoisomerase resulted in a 12-fold decrease in ATPase activity, comparable to that observed on both linear double-stranded (ds)DNA and (+)scDNA. In addition to the elevated activity in the presence of (-)scDNA, RecG also has high activity on model 4Y-substrates (i.e. chicken foot structures). This is due largely to the high apparent affinity of the enzyme for this DNA substrate, which is 46-fold higher than a 2Y-substrate (i.e. a three-way with two single-stranded (ss)DNA arms). Finally, the enzyme exhibited significant, but lower activity on ssDNA. This activity was enhanced by the Escherichia coli stranded DNA-binding protein (SSB) protein, which occurs through stabilizing of the binding of RecG to ssDNA. Stabilization is not afforded by the bacteriophage gene 32 protein, indicating a species specific, protein-protein interaction is involved. These results combine to provide significant insight into the manner and timing of the interaction of RecG with DNA at stalled replication forks.
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PMID:Characterization of the ATPase activity of the Escherichia coli RecG protein reveals that the preferred cofactor is negatively supercoiled DNA. 1729 98

The RecQ family of DNA helicases consists of specialized DNA unwinding enzymes that promote genomic stability through their participation in a number of cellular processes, including DNA replication, recombination, DNA damage signaling, and DNA repair pathways. Mutations resulting in the inactivation of some but not all members of the RecQ helicase family can lead to human syndromes which are characterized by marked chromosomal instability and an increased predisposition to cancer. An evolutionarily conserved interaction between RecQ helicases and topoisomerase 3s has been established, and this interaction is important in the regulation of recombination and genomic stability. Topoisomerases are critical in the cell because they relieve helical stress that arises when DNA is unwound. Topoisomerases function by breaking and rejoining DNA. By inhibition of the rejoining function, topoisomerase inhibitors are potent chemotherapeutic agents that have been used successfully in the treatment of hematologic malignancies and other cancers. This review discusses the roles of RecQ helicases in genomic stability, the interplay between RecQ helicases and topoisomerase 3s, and current and future prospects for targeting these interactions to develop novel anticancer therapies.
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PMID:The broken genome: genetic and pharmacologic approaches to breaking DNA. 1745 18


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