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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.
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PMID:Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus. 6 60

Polynucleotide templates were copied by avian myeloblastosis virus DNA polymerase ("reverse transcriptase") and the frequency and distribution of errors were determined. The error rate with [r(pA)2500-d(pT)12-18] template-initiator under a variety of conditions was approximately 1/600, i.e. one incorrect dCMP incorporated for 600 correct dTMP polymerized. Addition of the metal chelator o-phenanthroline to the reaction inhibited the incorporation of correct and incorrect nucleotides proportionately. The enzyme inhibited a pH optimum of 8.5 and the error rate remained constant over a range of pH (6.0 to 10.0). The rate of polymerization was greater at higher temperatures and approximately doubled for every 10 degrees increase. The error rate was constant at all temperatures. These results indicate that the purified avian myeloblastosis virus DNA polymerase synthesizes polydeoxynucleotides with an unusually large number of errors in base-pairing. Velocity sedimentation of the products of the reaction obtained at various times during the course of synthesis indicate that: (a) the entire length of the 14 S template was copied, and (b) the incorporation of the incorrect nucleotide did not terminate chain propagation. Isopyknic banding in neutral and alkaline cesium sulfate gradients showed that the noncomplementary nucleotides are incorporated into the polydeoxynucleotide product. Stepwise degradation and nearest neighbor analysis of the reaction product indicated that (a) the correct and incorrect nucleotides are present in phosphodiester linkages, (b) the errors are not concentrated at either termini; and (c) the errors are uniformly distributed throughout the newly synthesized polydeoxynucleotide.
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PMID:On the fidelity of DNA replication. Characterization of polynucleotides with errors in base-pairing synthesized by avian myeloblastosis virus deoxyribonucleic acid polymerase. 16 92

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.
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PMID:Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides. 17 98

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.
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PMID:Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA. 34 4

The alternating copolymer poly(dC-dG) has been methylated with either dimethyl sulphate or N-methyl-N-nitrosourea and the levels of the various methylation products determined. In addition to the 3-methylcytosine, 3-methylguanine and 7-methylguanine (produced by both agents) reaction with N-methyl-N-nitrosourea also yielded easily detectable amounts of O(6)-methylguanine and phosphotriesters. These methylated polymers were then used as templates in an in vitro assay with Escherichia coli DNA polymerase I measuring the incorporation of complementary (dCMP and dGMP) and noncomplementary (dAMP and dTMP) nucleotides. When the dimethyl sulphate-methylated polymer was used as template there was virtually no detectable incorporation of non-complementary nucleotides indicating that no miscoding could be attributed to the presence of 3-methylcytosine, 3-methylguanine or 7-methylguanine. However, when the N-methyl-N-nitrosourea-methylated polymer was used as template there was a specific incorporation of dTMP but not of dAMP. The amount of dTMP incorporated was always less than the level of O(6)-methylguanine in the template and was found to vary with the relative concentrations of the deoxynucleoside 5'-triphosphates in the assay. As the amount of dCTP present in the assay was decreased the wrong incorporation of dTMP increased and approached the level that would have been expected for a one-to-one miscoding by O(6)-methylguanine as the concentration of dCTP approached zero. The results indicate that O(6)-methylguanine is capable of miscoding with a DNA polymerase but the miscoding is competitive with the normal incorporation of dCMP: when the 5'-triphosphate precursors are present in equal amounts approximately one O(6)-methylguanine in three miscodes leading to the incorporation of dTMP.
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PMID:DNA synthesis with methylated poly(dC-dG) templates. Evidence for a competitive nature to miscoding by O(6)-methylguanine. 37 5

Some mutations in the structural gene for T4 DNA polymerase (gene 43) behave as suppressors of a deficiency in T4 dCMP-hydroxymethylase (gene 42). The suppression appears to involve a functional interaction between the two enzymes at the level of DNA replication. The hydroxymethylase deficiency caused DNA structural abnormalities in replication, and DNA polymerase lesions appeared to partially reverse these abnormalities. The results do not necessarily imply protein-protein interactions between the two enzymes, although both enzymes appear to play roles in controlling the fidelity of phage DNA replication.
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PMID:In vivo functional interaction between DNA polymerase and dCMP-hydroxymethylase of bacteriophage T4. 56 23

The labelling of mouse DNA by nick translation with DNA polymerase I has been investigated with respect to the time of incubation, requirement for DNAase I, size of the product, and uniformity of labelling, and the hybridisability and stability of the resultant labelled probes. Total mouse DNA and reannealed unique mouse DNA sequences can be labelled by nick translation in the presence of [3H]dCTP and [3H]TTP to a specific activity of 7 . 10(6)--20 . 10(6) cpm/microgram DNA. The hybridisation characteristics of nick-translated whole DNA with an excess of unlabelled mouse-embryo driver DNA indicates that no preferential labelling of repetitive or unique DNA sequence classes occurs. In addition, the proportion of unique DNA sequences labelled by nick translation which hybridises with polyadenylated nuclear RNA from Friend cells is the same as that of unique DNA sequences isolated from cells labelled with [3H]thymidine in vivo, indicating that few (if any) of the unique DNA sequences are unrepresented in the nick-translated probe. Probes which contain [3H]dTMP are unstable, and show a considerable reduction in hybridisability over a period of 6 months at --20 degrees C. The decrease is accompanied by an increase in the number of mismatched sites in duplexes containing the labelled probe (as shown by thermal stability measurements of hybrid molecules) and a decrease in the rate of hybridisation of the probe with total mouse DNA. In contrast, DNA which is labelled with [3H]dCMP alone is stable, and does not show any decrease in hybridisability on prolonged storage.
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PMID:Nick translation of mammalian DNA. 57 Apr 19

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.
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PMID:On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase. 86 97

In earlier reports we have suggested that bacteriophate T4 DNA replication occurs in a complex composed of the proteins required for polymerization and the system of enzymes synthesizing the deoxyribonucleoside triphosphate precursors of DNA. T4-induced dCMP hydroxymethylase and dTMP synthetase, though demonstrable in extracts soon after infection, are not active in vivo until about 5 min. The in vivo activities increase exponentially for approximately 15 min and then become constant. We have suggested that the exponential period represents the formation of the complexes. This paper shows that the initiation of DNA synthesis and of the two deoxyribonucleotide-synthesizing activities occurs simultaneously and with coinciding exponential kinetics. The in vivo activities of the two enzymes were tested after infection by a number of T4 amber Dna- mutants. Their activities were essentially unchanged compared to the wild-type phage, except on infection by mutants of gene 43 (T4 DNA nucleotidyltransferase or DNA polymerase). With these mutants the rate of increase of dTMP synthetase and dCMP hydroxymethylase activities was always substantially lower than after infection by wild-type phage. It is proposed that an intimate interaction occurs between T4-induced DNA polymerase and the complex of enzymes forming 5-hydroxymethyl-dCMP and dTMP.
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PMID:Simultaneous initiation of synthesis of bacteriophage T4 DNA and of deoxyribonucleotides. 106 86

During the fractionation of various enzymes concerned with DNA synthesis from the postmicrosomal supernatant fraction of various tissues, DNA polymerace [EC 2.7.7.7], thymidine kinase [EC 2.7.1.75], dTMP kinase [EC 2.7.4.9], deoxycytidine kinase [EC 2.7.1.74], and deoxycytidine monophosphokinase (dCMP kinase) [EC 2.7.4.14] were found in the pellet fraction of postmicrosomal supernatant. Further, the uridine kinase [EC 2.7.1.48] and aspartate transcarbamylase [EC 2.1.3.2] activities of postmicrosomal supernatant from various tissues were also present in this pellet fraction. The activities of DNA polymerase, thymidine kinase, uridine kinase, and aspartate transcarbamylase from normal and regenerating rat liver, and Yoshida sarcoma were higher in the pellet fraction than in the supernatant. On the other hand, the activities of dTMP kinase, dCMP kinase, and orotidine-5'-phosphate decarboxylase [EC 4.1.1.23] were lower in the pellet fraction than in the supernatant. The pellet fractions of regenerating rat liver and Yoshida sarcoma showed a remarkable incorporation of various precursors (thymidine, dTMP, deoxycytidine, and dCMP) into DNA in the presence of a suitable DNA template, ATP and all four deoxynucleoside 5'-triphosphates for DNA synthesis. Normal adult rat liver catalyzed a much smaller incorporation of all these precursors, except for dCMP.
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PMID:Intracellular distribution of various enzymes concerned with DNA synthesis from normal and regenerating rat liver, and Yoshida sarcoma. 113 86


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