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)

The RecQ helicase superfamily has been implicated in DNA repair and recombination. At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5. Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability. RecQ5 is small, containing only a core part of the RecQ helicase, but three isomer transcripts code for small RecQ5alpha (corresponding to the original RecQ5 with 410 amino acids), new large RecQ5beta (991 amino acids) and small RecQ5gamma (435 amino acids) proteins that contain the core helicase motifs. By determining the genomic structure, we found that the three isoforms are generated by differential splicing from the RecQ5 gene that contains at least 19 exons. Northern blot analysis using a RecQ5beta-specific probe indicates that RecQ5beta mRNA is expressed strongly in the testis. Immunocytochemical staining of three N-terminally tagged RecQ5 isomers expressed in 293EBNA cells showed that RecQ5beta migrates to the nucleus and exists exclusively in the nucleoplasm, while the small RecQ5alpha and RecQ5gamma proteins stay in the cytoplasm. Immunoprecipitation and an extended cytochemical experiment suggested that the nucleoplasmic RecQ5beta, like yeast Sgs1 DNA helicase, binds to topoisomerases 3alpha and 3beta, but not to topoisomerase 1. These results predict that RecQ5beta may have an important role in DNA metabolism and may also be related to a distinct genetic disease.
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PMID:Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta. 1071 Apr 32

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.
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PMID:Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle. 1072 63

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.
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PMID:Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling. 1074 89

Quinolone antibacterial drugs target both DNA gyrase (Gyr) and topoisomerase IV (Topo IV) and form topoisomerase-quinolone-DNA ternary complexes. The formation of ternary complexes results in the inhibition of DNA replication and leads to the generation of double-strand breaks and subsequent cell death. Here, we have studied the consequences of collisions between the UvrD helicase and the ternary complexes formed with either Gyr, Topo IV, or a mutant Gyr, Gyr (A59), which does not wrap the DNA strand around itself. We show (i) that Gyr-norfloxacin (Norf)-DNA and Topo IV-Norf-DNA, but not Gyr (A59)-Norf-DNA, ternary complexes inhibit the UvrD-catalyzed strand-displacement activity, (ii) that a single-strand break is generated at small portions of the ternary complexes upon their collisions with UvrD, and (iii) that the majority of Topo IV-Norf-DNA ternary complexes become nonreversible when UvrD collides with the Topo IV-Norf-DNA ternary complexes, whereas the majority of Gyr-Norf-DNA ternary complexes remain reversible after their collision with the UvrD helicase. These results indicated that different DNA repair mechanisms might be involved in the repair of Gyr-Norf-DNA and Topo IV-Norf-DNA ternary complexes.
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PMID:Distinct effects of the UvrD helicase on topoisomerase-quinolone-DNA ternary complexes. 1079 52

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.
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PMID:Genetic analysis of the Saccharomyces cerevisiae Sgs1 helicase defines an essential function for the Sgs1-Top3 complex in the absence of SRS2 or TOP1. 1101 37

Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis.
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PMID:Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage. 1108 95

Some topoisomerase inhibitors trap covalent topoisomerase-DNA complexes as topoisomerase-drug-DNA ternary complexes. Ternary complex formation results in inhibition of DNA replication and generation of permanent double-strand breaks. Recent demonstrations of the stimulation of covalent topoisomerase-DNA complex formation by DNA lesions suggest that DNA damage may act as an endogenous topoisomerase poison. We have investigated the effects of abasic (AP) sites on topoisomerase IV (Topo IV). AP sites can stimulate the formation of covalent Topo IV-DNA complexes when they are located either within the 4 base overhang generated by DNA scission or immediately 5' to the point of scission (the -1 position). Thus, the AP site acts as a position-specific, endogenous topoisomerase poison. Both EDTA and salt can reverse covalent Topo IV-DNA complexes induced by AP sites located within the 4 base overhang. Interestingly, an AP site at the -1 position inhibits EDTA-mediated reversal of formation of the covalent Topo IV-DNA complex. Furthermore, we find that, unlike quinolone-induced covalent Topo IV-DNA complexes, AP site-induced covalent Topo IV-DNA complexes do not inhibit the helicase activities of the DnaB and T7 Gene 4 proteins. These results suggest that the AP site-induced poisoning of Topo IV does not arrest replication fork progression.
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PMID:Replicative helicases can translocate through abasic site-induced covalent topoisomerase IV-DNA complexes. 1116 Aug 81

Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.
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PMID:Bacteriophage T4 proteins replicate plasmids with a preformed R loop at the T4 ori(uvsY) replication origin in vitro. 1117 9

Deficiency in a helicase of the RecQ family is found in at least three human genetic disorders associated with cancer predisposition and/or premature ageing. The RecQ helicases encoded by the BLM, WRN and RECQ4 genes are defective in Bloom's, Werner's and Rothmund-Thomson syndromes, respectively. Cells derived from individuals with these disorders in each case show inherent genomic instability. Recent studies have demonstrated direct interactions between these RecQ helicases and human nuclear proteins required for several aspects of chromosome maintenance, including p53, BRCA1, topoisomerase III, replication protein A and DNA polymerase delta. Here, we review this network of protein interactions, and the clues that they present regarding the potential roles of RecQ family members in DNA repair, replication and/or recombination pathways.
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PMID:DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders. 1125 7

The Saccharomyces cerevisiae SGS1 gene is a member of the RecQ family of ATP-dependent DNA helicases, which includes the human WRN, BLM and RECQ4 genes. Mutations in the WRN gene cause the human premature ageing disorder, Werner's syndrome. Deletion of the SGS1 gene also causes premature ageing in yeast, suggesting that the molecular mechanisms of cellular ageing may be evolutionarily conserved. To investigate the role of the RecQ helicase domain in ageing, a point mutation (SGS1 K(706)-->A) known to eliminate the DNA helicase activity of Sgs1p was constructed. This mutant allele failed to rescue the premature ageing of the sgs1Delta strain, demonstrating that Sgs1p DNA helicase activity is required for a normal lifespan. In contrast, the SGS1 K(706)-->A allele was sufficient to rescue the hypersensitivity of the sgs1Delta strain to topoisomerase inhibitors, but not other genotoxic agents. These findings support the idea that Sgs1p fulfils multiple cellular functions, and that DNA helicase activity is dispensable for some of these (e.g. functional interaction with topoisomerases), but essential for others (e.g. longevity).
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PMID:The DNA helicase activity of yeast Sgs1p is essential for normal lifespan but not for resistance to topoisomerase inhibitors. 1138 27


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