Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [(3)H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. & Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I (polA(-)) and polA(-) strains that are also defective in uracil N-glycosidase, an enzyme required for the excision-repair of uracil in DNA (polA(-)ung(-)). Little or no difference was detected in the level of Okazaki fragments in the polA(-) strain as compared with the polA(-)ung(-) strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N-glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.
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PMID:Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome. 20 31

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.
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PMID:Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development. 169 67

Ten temperature-sensitive mutants of T4D have been examined to find the times in the lytic cycle at which a shift to a restrictive temperature no longer exerts a lethal effect. Nine of these mutants play a role in phage DNA synthesis. The results of these experiments suggest that the products of three of the genes tested-genes 42, 44, and 45-have control or complexing functions in addition to any enzymatic ones. The data also indicate that the products of genes 30 and 41 may act cooperatively. The properties of mutants of gene 43 (DNA polymerase) and gene 56 (dCTP-dUTPase) suggest that the production of a certain length of concatameric DNA is necessary before viable phage can be produced.
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PMID:Events occurring during the replication of bacteriophage T4 DNA. 489 86

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.
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PMID:In vitro synthesis of bacteriophage phi X174 by purified components. 622 17

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
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PMID:Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides. 675 52

Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.
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PMID:Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications. 832 30

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.
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PMID:Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. 838 67

After T4 bacteriophage infection of Escherichia coli, the enzymes of deoxyribonucleoside triphosphate biosynthesis form a multienzyme complex that we call T4 deoxyribonucleoside triphosphate (dNTP) synthetase. At least eight phage-coded enzymes and two enzymes of host origin are found in this 1.5-mDa complex. The complex may shuttle dNTPs to DNA replication sites, because replication draws from small pools, which are probably highly localized. Several specific protein-protein contacts within the complex are described in this paper. We have studied protein-protein interactions in the complex by immobilizing individual enzymes and identifying radiolabeled T4 proteins that are retained by columns of these respective affinity ligands. Elsewhere we have described interactions involving three T4 enzymes found in the complex. In this paper we describe similar analysis of five more proteins: dihydrofolate reductase, dCTPase-dUTPase, deoxyribonucleoside monophosphokinase, ribonucleotide reductase, and E. coli nucleoside diphosphokinase,. All eight proteins analyzed to date retain single-strand DNA-binding protein (gp32), the product of T4 gene 32. At least one T4 protein, thymidylate synthase, binds directly to gp32, as shown by affinity chromatographic analysis of the two purified proteins. Among its several roles, gp32 stabilizes single-strand template DNA ahead of a replicating DNA polymerase. Our data suggest a model in which dNTP synthetase complexes, probably more than one per growing DNA chain, are drawn to replication forks via their affinity for gp32 and hence are localized so as to produce dNTPs at their sites of utilization, immediately ahead of growing DNA 3' termini.
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PMID:T4 phage gene 32 protein as a candidate organizing factor for the deoxyribonucleoside triphosphate synthetase complex. 862 61

The purpose of this review is to summarize information published since 1990 on DNA replication, recombination and repair of vaccinia virus, a poxvirus. Temperature-sensitive mutations reveal four essential genes related to viral DNA replication: the E9L DNA polymerase, B1R protein kinase, D5R protein, and D4R uracil DNA glycosylase. Other proteins are likely to be also involved in viral DNA replication: the H6R DNA topoisomerase, I3L single stranded-DNA binding protein, H5R virosome-associated protein, and A50R DNA ligase. In addition, several viral-encoded proteins do regulate the level of the deoxyribonucleoside triphosphate pool: the J2R thymidine kinase, A48R thymidylate kinase, 14L and F4L subunits of ribonucleotide reductase, and F2L dUTPase. Despite the apparent simplicity of the mechanism of vaccinia virus DNA replication, several important questions related to the three Rs remain unsolved.
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PMID:Vaccinia virus DNA replication: a short review. 882 74

We report the nucleotide sequence of a 31-kb segment at the left genome end of bovine herpesvirus-1 (BHV-1) and show that it comprises 19 different open reading frames (ORFs), including seven which have been described previously (circ, dUTPase, UL49.5, alpha TIF, VP8, glycoprotein C, and ribonucleotide reductase small subunit). The new sequence resulted in a correction at the C-terminus of glycoprotein C. All 19 ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 (HSV-1) in the range of the UL54 to UL37 genes. No BHV-1 homologs of the HSV-1 UL56, UL55, and UL45 genes were identified. The BHV-1 circ gene was the only gene without a HSV-1 counterpart. The additional ORFs 1 and 2 found at the left genome end of equine herpesvirus-1 (EHV-1) were absent in BHV-1. Among the newly sequenced BHV-1 ORFs are homologs of ICP27 (UL54), glycoprotein K (UL53), helicase-primase (UL52), DNA polymerase accessory protein (UL42), ribonucleotide reductase large subunit (UL39), and several virion proteins (UL49, UL46, UL43, UL41, UL38, UL37), most of which are strongly conserved in all herpesviruses. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed.
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PMID:Gene contents in a 31-kb segment at the left genome end of bovine herpesvirus-1. 901 Sep 99


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