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:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
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PMID:A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro. 216 68

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.
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PMID:Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. 240 71

The replication of plasmid pBR322 DNA has been reconstituted with purified proteins from Escherichia coli. Initiation of the leading-strand requires RNA polymerase holoenzyme, DNA polymerase I, RNase H, and DNA gyrase. Initiation of the lagging-strand requires the primosomal proteins (the dnaB, dnaC, and dnaG proteins, replication factor Y (protein n') and proteins i, n, and n") and the single-stranded DNA binding protein. DNA polymerase III holoenzyme is required for extensive elongation of the nascent DNA chains. The products of this replication reaction are primarily nonsegregated daughter molecules. However, the addition of small amounts of soluble extract from E. coli results in the completion and segregation of these molecules to give mature form I DNA, suggesting that additional factors are required for this process. Topoisomerase I is necessary to make the replication system specific for pBR322 DNA as a template, indicating that the linking number of the DNA, determined by an equilibrium between the opposing activities of topoisomerase I and DNA gyrase, plays a crucial role in determining the reactivity of the DNA molecule toward initiating DNA replication. The function of the proteins involved in the replication of this closed-circular, double-stranded, superhelical DNA is discussed.
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PMID:Replication of pBR322 DNA in vitro with purified proteins. Requirement for topoisomerase I in the maintenance of template specificity. 299 Dec 40

Progress of the replication forks of the Escherichia coli chromosome depends on a multisubunit DNA polymerase (for chain elongation) and a primosome (for chain initiations), together comprising about 30 polypeptides with a mass in excess of 10(6) daltons. Integration of their actions with those of helicases and DNA binding proteins suggest a more complex and integrated replisome assembly with novel possibilities for concurrent replication of both parental strands. Initiation of a new cycle of chromosome replication at its unique 245-bp (oriC) is being studied in a soluble enzyme system with plasmids, autonomous replication of which depends on the oriC sequence. Required proteins include RNA polymerase, DNA gyrase, dnaA protein (with 4 strong binding sites in oriC), HU protein, and additional proteins (e.g., topoisomerase I and ribonuclease H) that confer oriC specificity by suppressing initiation of replication elsewhere on the duplex DNA. Clarification of the biochemical mechanisms of replication is fundamental for understanding cell growth and development. Knowledge of the biochemistry of initiating a cycle of chromosome replication opens the way toward exploring the regulation of the cell cycle. I remain faithful to the conviction that anything a cell can do, a biochemist should be able to do. He should do it even better, being freed from the constraints of substrate and enzyme concentrations, pH, ionic strength, and temperature, and by having the license to introduce novel reagents to drive or restrain a reaction. Put another way, one can be creative more easily with a reconstituted system. One can grapple directly with the molecules instead of trying by remote means to manipulate their structures or levels in the intact cell. Enzyme purification carries many dangers beyond the well-known exposure of the fragile enzyme to the hostilities of an unfamiliar environment, high dilution, glass containers and a denaturable investigator. But the rewards of enzyme purification have justified the effort. The polymerases, nucleases, ligases purified out of curiosity about the mechanisms of replication, repair and recombination have supplied the cast of actors responsible for the current drama of genetic engineering. Beyond the uses of these enzymes as reagents, understanding the mechanisms of DNA metabolism will have practical value in manipulating the replication of plasmids and viruses and the expression of their genes and, beyond that, in obtaining a more secure grasp of chromosome structure and function.
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PMID:Enzyme studies of replication of the Escherichia coli chromosome. 609 56

Crude soluble enzyme fractions that initiate bidirectional replication from the unique Escherichia coli chromosomal origin (oriC) have been fractionated further to identify the components and mechanisms of this complex system. Among the necessary factors is a class of specificity proteins that suppress initiations on plasmids which lack the oriC sequence and which do not depend on dnaA protein. One such specificity factor has been identified as RNase H (Ogawa, T., Pickett, G. G., Kogoma, T., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1040-1044). Another, described here, has proved to be topoisomerase I. A protein was purified to near homogeneity based on assays of (i) inhibition of the replication of plasmids (and other supercoiled DNA) lacking oriC and (ii) conferral of dnaA protein dependence on the replication of an oriC plasmid. This specificity protein is indistinguishable from authentic E. coli topoisomerase I by several criteria: (i) molecular weight under denaturing conditions, (ii) relaxation activity on negatively supercoiled DNA, (iii) cleavage pattern of single-stranded DNA, (iv) specificity factor activity, and (v) neutralization of activity by antibody against topoisomerase I. One possible mechanism of the specificity action of topoisomerase I is destabilization of primers for replication except when they are preserved at an oriC sequence bound by dnaA protein and other replication proteins.
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PMID:Topoisomerase I confers specificity in enzymatic replication of the Escherichia coli chromosomal origin. 633 Jan 14

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.
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PMID:Further purification and characterization of a multienzyme complex for DNA synthesis in human cells. 830 Jul 57

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

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.
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PMID:Prominent mitochondrial DNA recombination intermediates in human heart muscle. 1171 92

Acid resistance (AR) in Escherichia coli is important for its survival in the human gastrointestinal tract and involves three systems. The first AR system is dependent on the sigma factor RpoS. The second system (the GAD system) requires the glutamate decarboxylase isoforms encoded by the gadA and gadB genes. The third system (the ARG system) requires the arginine decarboxylase encoded by adiA. Loss of topoisomerase I function from topA deletion or Tn10 insertion mutations lowered the resistance to killing by pH 2 or 2.5 treatment by 10-fold to >100-fold. The RpoS and GAD systems were both affected by the topA mutation, but the ARG system of AR was not affected. Northern blot analysis showed that induction of gadA and gadB transcription in stationary phase and at pH 5.5 was decreased in the topA mutant. Western blot analysis showed that the topA mutation did not affect accumulation of RpoS, GadX or GadW proteins. Topoisomerase I might have a direct influence on the transcription of AR genes. This influence does not involve R-loop formation as the overexpression of RNase H did not alleviate the decrease of AR caused by the topA mutation. The effect of the topA mutation could be suppressed by an hns mutation, so topoisomerase I might be required to counteract the effect of H-NS protein on gene expression, in addition to its influence on RpoS-dependent transcription.
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PMID:Loss of topoisomerase I function affects the RpoS-dependent and GAD systems of acid resistance in Escherichia coli. 1607 54

Alterations of RNA sequences and structures, such as those from editing and alternative splicing, result in two or more RNA transcripts from a DNA template. It was thought that in yeast, RNA editing only occurs in tRNAs. Here, we found that Saccharomyces cerevisiae have all 12 types of RNA-DNA sequence differences (RDDs) in the mRNA. We showed these sequence differences are propagated to proteins, as we identified peptides encoded by the RNA sequences in addition to those by the DNA sequences at RDD sites. RDDs are significantly enriched at regions with R-loops. A screen of yeast mutants showed that RDD formation is affected by mutations in genes regulating R-loops. Loss-of-function mutations in ribonuclease H, senataxin, and topoisomerase I that resolve RNA-DNA hybrids lead to increases in RDD frequency. Our results demonstrate that RDD is a conserved process that diversifies transcriptomes and proteomes and provide a mechanistic link between R-loops and RDDs.
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PMID:RNA-DNA sequence differences in Saccharomyces cerevisiae. 2763 43


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