Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg2+ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3' to 5' exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.
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PMID:A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus. 630 34

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.
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PMID:The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria. 630 36

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.
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PMID:Stimulation of DNA synthesis by mouse DNA helicase B in a DNA replication system containing eukaryotic replication origins. 779 3

We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.
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PMID:DNA replication from initiation zones of mammalian cells in a model system. 793 72

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.
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PMID:Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC. 819 75

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.
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PMID:Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice. 850 71

The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells. A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth. Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS. The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced. Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S. typhimurium cells by the macrophage. These data indicate that DNA topology in S. typhimurium undergoes significant changes during adaptation to the intracellular environment. A model describing how this process may operate is discussed.
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PMID:DNA topology and adaptation of Salmonella typhimurium to an intracellular environment. 1087 30

The simian virus 40 large tumour-antigen (T antigen) DNA helicase is a hexameric structure; it has been proposed that, in viral DNA replication, two of these hexamers are combined to form a bipartite holoenzyme that acts concurrently at both forks of a replication bubble. In a search for structural components of this helicase complex, we have identified nucleolin as a specific binding protein for the T-antigen hexamer. We show that nucleolin, in co-operation with human topoisomerase I, mediates the cohesion of the T-antigen helicase holoenzyme during plasmid unwinding. Our results provide biochemical evidence for a direct role of nucleolin in DNA replication, in addition to its known function in ribosome biogenesis. The data presented here suggest that nucleolin enables the formation of a functional 'helicase-swivelase' complex at the replication fork.
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PMID:Bidirectional DNA unwinding by a ternary complex of T antigen, nucleolin and topoisomerase I. 1263 43

The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral single-strand DNA-binding protein promotes assimilation of single-stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral single-strand DNA-binding protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.
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PMID:Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1. 1292 2

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.
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PMID:Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication. 1502 9


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