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)

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.
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PMID:Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I. 634 14

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.
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PMID:Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro. 634 19

The synthetic DNA polymers, poly(dG-dC), poly(dC), poly(dA-dT), poly(dA) and poly(dT), were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS) and UV irradiation. The modified polymers were used as templates to examine the incorporation of non-complementary nucleotides by E. coli DNA polymerase I. Methylation of poly(dG-dC) by MNNG predominantly induced the misincorporation of dTMP, whereas methylation by MMS induced that of dAMP. Treatment of poly(dT) with MNNG caused the misincorporation of dGMP to a considerable extent, but MMS did not enhance the error on poly(dT). The misincorporation of dAMP on poly(dC) and that of dGMP on poly(dA) were also increased by these chemicals. UV irradiation of poly(dT) and poly(dC) induced the error of dGMP and dAMP, respectively. These data on MNNG and MMS in vitro were in fair agreement with the directions of mutation in vivo. But the predominant induction of transitions by UV in vitro did not agree with the UV-induced transversions in E. coli. This inconsistency suggested the participation of other factors than direct mispairing in UV-induced transversion. Modification of DNA polymerase I by MNNG changed the ratio of polymerase to 3' leads to 5' exonuclease activity altering the fidelity of this enzyme, whereas MMS and UV-irradiation did not alter the fidelity of the enzyme.
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PMID:Misincorporation in DNA synthesis after modification of template or polymerase by MNNG, MMS and UV radiation. 634 75

M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
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PMID:Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. 635 60

The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than T4 and T5 DNA polymerases. At the same time, there was a higher turnover of dCTP than of dTTP by the E. coli enzyme. However, in all cases, the turnover of deoxynucleotides during replication of m6Gua-containing templates was more than that observed with templates without the alkylated base.
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PMID:Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication. 637 99

This paper describes techniques and strategies for semi-preparative high-performance liquid chromatographic (HPLC) purification of 2'-deoxynucleoside 5'-triphosphates (dNTPs). The procedure yields dNTPs that are sufficiently pure for use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. Anion-exchange HPLC was used to purify the four normal dNTPs (dATP, dGTP, dCTP and dTTP), plus the chemically modified analogues, 5-BrdUTP, 5-IodUTP and 1,N6-etheno-dATP (epsilon dATP). Baseline separations were achieved by isocratic elution of dNTPs with potassium dihydrogen phosphate mobile phase. In general, the resolution of dNTPs was highly dependent on pH, although the influence of mobile phase composition on separation of dNTPs was not the same for all three HPLC packing materials used. A Hewlett-Packard diode array detector was extremely valuable in the identification of contaminating peaks and in the development of optimal mobile phase conditions for dNTP purification. The pure dNTPs were used in the electrophoretic assay of misincorporation, yielding information about the mispairing potential of the modified dNTPs. BrdUMP and IodUMP were misincorporated in place of dCMP during chain elongation catalyzed by purified DNA polymerase I of Escherichia coli. epsilon dAMP was incorporated into DNA in place of dAMP, although at much lower efficiency than dAMP.
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PMID:High-performance liquid chromatographic purification of deoxynucleoside 5'-triphosphates and their use in a sensitive electrophoretic assay of misincorporation during DNA synthesis. 639 78

To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in phi29 DNA replication, extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex. Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex, suggesting that these proteins are not involved in the initiation of phi29 DNA replication. ddATP or aphidicolin, inhibitors of DNA chain elongation, had also no effect on the formation of the initiation complex.
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PMID:Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex. 640 61

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.
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PMID:A novel DNA polymerase induced by Bacillus subtilis phage phi 29. 642 95

Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3.
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PMID:Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29. 642 20

The phage phi 29 protein p2, required for the formation of the protein p3-dAMP initiation complex, has been purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of molecular weight 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when phi 29 DNA-protein p3 was used as template. In addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 mutant sus2-infected Bacillus subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of phi 29 DNA-protein p3 as template. However, when purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the phi 29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage phi 29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.
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PMID:Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication. 643 48


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