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 replication of simian virus 40 origin-containing DNA has been reconstituted in vitro with SV40 large T antigen and purified proteins isolated from HeLa cells. Covalently closed circular DNA (RF I') daughter molecules are formed in the presence of T antigen, a single-stranded DNA binding protein and DNA polymerase alpha-primase complex, together with ribonuclease H, DNA ligase, topoisomerase II, and a double-stranded specific exonuclease that has been purified to homogeneity. The 44-kDa exonuclease-digested oligo(rA) annealed to poly(dT) in the 5'----3' direction. DNA ligase and the 5'----3' exonuclease were essential for RF I' formation. Covalently closed circular duplex DNA and full length linear single-stranded DNA were detected by alkaline gel electrophoresis as products of the complete system. DNA replication in the absence of either DNA ligase or the 5'----3' exonuclease yielded DNA products that were half length (approximately 1500 nucleotides) and smaller Okazaki-like fragments (approximately 200 nucleotides). Hybridization experiments showed that the longer chains were synthesized from the leading strand template, while the small products were synthesized from the lagging strand template. These results suggest that the RNA primers attached to 5' ends of replicated DNA are completely removed by the 5'----3' exonuclease, with the assistance of RNase H.
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PMID:Complete enzymatic synthesis of DNA containing the SV40 origin of replication. 284 39

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.
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PMID:Processivity of the DNA polymerase alpha-primase complex from calf thymus. 360 95

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.
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PMID:Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. 753 35

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
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PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40

Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.
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PMID:Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes. 880 83

Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.
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PMID:DNA topoisomerases regulate R-loop formation during transcription of the rrnB operon in Escherichia coli. 913 42

One major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop. The goal of the present study was to identify other potential regulators of R-loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R-loop formation. We show that DNA topoisomerase III is able to relax transcription-induced negative supercoiling both in vitro and in vivo. An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loop-dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.
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PMID:Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli. 1063 77

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.
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PMID:SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. 1098 59

The structures of the catalytic core of two HIV-1 encoded enzymes play a crucial role in the retroviral cycle: integrase and RNase H exhibit striking similarities. These enzymes also share a similar mechanism of catalysis. The homologies between RNase H and integrase led to studying the effect of the RNase H inhibitors on integrase. ODNs aptamers active on RNase H were shown to be strong IN inhibitors. On the contrary, compounds from the diketo acid family were previously known as integrase inhibitors. One compound of this family is able to inhibit the RNase H activity, but has no effect on integrase. Cellular topoisomerase 1 also shares a mechanism similar to that of HIV-1 integrase and RNase H. It has been reported to be present in retroviral particles and to enhance cDNA synthesis. Some topoisomerase inhibitors have been shown to be active on integrase. Moreover, topoisomerase, integrase and RNase H are inhibited by G-rich oligonucleotides. A G-quartet structure is necessary for integrase, but not for topoisomerase inhibition. This suggests that prototype structures can be exploited to develop inhibitors of two related enzymes, such as the RNase H and integrase activities of HIV-1 RT.
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PMID:Closely related antiretroviral agents as inhibitors of two HIV-1 enzymes, ribonuclease H and integrase: "killing two birds with one stone". 1557 66


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