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)

phi 29 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The phi 29 DNA polymerase was purified from phage-infected cells by using poly(dA) X p(dT)12-18 as an assay template. The purified polymerase has an apparent molecular mass of 68 kDa in its native form and it appears to function as a monomer. The terminal protein was purified to homogeneity from Escherichia coli cells harboring a cloned plasmid that contained a phi 29 gene 3 segment. The molecular mass of the purified terminal protein was about 30 kDa in both the denatured and the native form. The protein apparently functions as a monomer. When the terminal protein and DNA polymerase were incubated in the presence of dATP, Mg2+, and phi 29 DNA-protein as template, the terminal protein bound covalently to dAMP. This reaction did not require ATP. In addition, these two purified fractions catalyzed DNA chain elongation from both ends of phi 29 DNA, yielding the expected 9- to 12-base fragment when assayed in the presence of 2',3'-dideoxycytidine triphosphate. These results indicate that phi 29 DNA polymerase catalyzes formation of the terminal protein-dAMP complex and can also catalyze chain elongation at least 9-12 bases from both ends of phi 29 DNA.
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PMID:Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase. 643 49

Heat treatment of poly(deoxycytidylic acid)-[poly(dC)] induces the formation of dUMP residues, which code for dAMP when replicated by Escherichia coli DNA polymerases I and III. The specificity of dUMP coding properties is indicated by the quantitative relation between the dAMP incorporated and the frequency of dUMP residues in the heat-treated poly(dC). The dAMP incorporation is prevented by preincubation of uracil containing poly(dC) with uracil-DNA glycosylase. The excision of uracil by uracil-DNA glycosylase leads to the formation of apyrimidinic sites (AP sites), which are barely replicated in vitro under physiological conditions. However, the alteration of E. coli DNA polymerase I fidelity of replication by Mn2+ greatly stimulates the replication of AP sites. There is a preferential incorporation of dAMP, as compared to dTMP, opposite the AP sites. The dAMP incorporation is prevented by preincubation of poly(dC) containing AP sites with Micrococcus luteus AP endonuclease B. The results show a close association between DNA repair by base excision and the prevention of mutagenic processes in vitro. Furthermore, since the alteration of DNA polymerase fidelity allows some replication of the noncoding DNA lesion (AP site), this could imply a role in SOS-induced mutagenesis in vivo.
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PMID:Coding properties of poly(deoxycytidylic acid) templates containing uracil or apyrimidinic sites: in vitro modulation of mutagenesis by deoxyribonucleic acid repair enzymes. 676 Aug 93

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.
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PMID:Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin. 678 51

Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation.
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PMID:Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism. 679 95

After treatment of poly(dC) by the simple alkylating agent (3H)dimethylsulfate, 90 per cent of the radioactivity cochromatographied with 3-methylcytosine and 10 per cent with 5-methylcytosine which is the normally occurring methylated base. In order to study the influence of 3-methylcytosine on DNA replication, untreated and DMS-treated poly(dC) were used as templates for E. coli DNA polymerase I. The alkylation of poly(dC) inhibits DNA chain elongation, and does not induce any mispairing under high fidelity conditions. The alteration of DNA polymerase I fidelity by manganese ions allows some replication of 3-methylcytosine which mispairs with either dAMP or dTMP. Our results suggest that 3-methylcytosine could be responsible, at least partially, for the killing and the mutagenesis observed after cell treatment by alkylating agents.
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PMID:Mutagenesis by alkylating agents: coding properties for DNA polymerase of poly (dC) template containing 3-methylcytosine. 681 12

The effects of manganese on DNA synthesis fidelity are measured using T4 DNA polymerase. When the nucleotide analogue 2-aminopurine deoxyribonucleoside triphosphate competes against dATP at thymine sites on template DNA, the aminopurine misincorporation frequency increases from 6.3% in the presence of Mg2+ to 29.2% in the presence of Mn2+. The major cause of the increased error rate is an approximate 4-fold increase in the frequency of aminopurine misinsertions. Exonucleolytic proofreading of aminopurine is similar in the presence of Mn2+ and Mg2+. However, the excision frequency of the correct nucleotide, dAMP, is increased 2-fold with Mn2+. In experiments in which insertion and incorporation velocities of aminopurine and adenine are measured independently of each other, a 5- to 10-fold decrease in the Michaelis constant for aminopurine is observed in the presence of Mn2+ compared to a 2-fold decrease in the Km for adenine. In contrast to the marked differential reduction in the ratio of aminopurine to adenine Km values, the maximum insertion velocities of both nucleotides are reduced by similar amounts (40-fold). We suggest that the mutagenic action of Mn2+ can be attributed primarily to a significant differential increase in binding of mispaired relative to correctly paired nucleotides to the polymerase-template complex. The resulting increase in the ratio of residence times for mispaired compared with correctly paired nucleotides on the complex results in their increased frequency of misinsertion. A smaller contributing factor to Mn2+-induced mutagenesis is a loss of proofreading specificity. We propose that the losses in both the specificities of nucleotide insertion and excision (proofreading) share a common molecular origin in which nucleotides are bound in the presence of Mn2+ in distorted configurations at the polymerase insertion and excision active sites resulting in increased nonspecific enzyme-substrate binding forces at the expense of template-substrate base pair specific hydrogen bonds.
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PMID:On the enzymatic basis for mutagenesis by manganese. 683 10

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase alpha and beta of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n-alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [3H]dAMP or [3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, alpha-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n-alkyl-dUTPs than beta-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2-14C]isopropyl-dUTP.
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PMID:A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2'-deoxyuridine 5'-triphosphates. 698 14

Chloroacetaldehyde, a rearranged metabolic product of the human carcinogen vinyl chloride, reacts with the DNA-like polymers poly(dA-dT) and poly(dC-dG) to form etheno-adducts of the adenine and cytosine bases. These treated polymers, when used as templates for E. coli DNA polymerase I in an in vitro assay, show a decreased ability to direct DNA synthesis. At the same time, increased relative levels of non-complementary nucleotides are incorporated. With the poly(dA-dT) templates 1 dGMP residue is incorporated for every approx 60 ethenoadenine residues present whilst no increased misincorporation of dCMP was detected. With the poly(dC-dG) templates 1 misincorporation of dAMP or dTMP occurred in the presence of approx 30 and 80 ethenocytosine residues respectively. A nearest neighbour analysis shows that with the modified poly(dC-dG) templates the majority of the errors were incorporated opposite cytosine (or modified cytosine) bases.
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PMID:The induction of errors during in vitro DNA synthesis following chloroacetaldehyde-treatment of poly(dA-dT) and poly(dC-dG) templates. 702 22

A detailed study of the mechanism of nascent chain elongation and of steady state kinetics of purified mouse DNA polymerase alpha has been conducted. Polymerization was examined using a model replication system of poly(dT) as template, oligo(rA) as primer, and dATP as nucleotide substrate, and the probability of chain termination was determined by measurement of the precise chainlength of the products. Reactions were conducted under conditions where products were not utilized as primer. Product chainlength analysis indicated that alpha-polymerase acted in a processive fashion, elongating the primer by the stepwise addition of up to 20 dAMP residues before dissociating. The probability of termination after each dAMP addition depended upon the chainlength of the product and upon the presence of several agents; spermine, spermidine, putrescine, nalidixic acid, or PPi caused a marked increase in termination after the first dAMP addition, and conversely, mouse helix destabilizing protein-1 caused the enzyme to continue extending the same product chain until 18 to approximately 35 dAMP residues had been added. From these and other data, it is concluded that the kinetic mechanisms of termination after the first dAMP addition and after subsequent dAMP additions are different. With this information on how alpha-polymerase elongates a nascent primer(dA)n molecule, a kinetic model and appropriate steady state rate equations were obtained for analysis of substrate initial velocity data and termination probabilities. The substrate kinetic patterns and PPi product inhibition results were consistent with the ordered Ter Ter mechanism Bi Uni Uni Bi Ping Pong proposed in the model, and the model also permits a rational explanation for the differences in termination probability and for the fact that substrate initial velocity plots were linear even though multiple residues of dATP combined with the enzyme during each catalytic cycle. In addition, the results suggest that a rate-limiting step in the steady state occurs at the transition between initiation and elongation, and that higher levels of template.primer increase the rate of this step. This secondary effect of template.primer is discussed in relation to other polymer-forming enzymes, and various kinetic mechanisms which require the presence of two template.primer-binding sites, effector and catalytic, are discussed for their fit to the experimental data.
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PMID:Studies on the mechanism of DNA polymerase alpha. Nascent chain elongation, steady state kinetics, and the initiation phase of DNA synthesis. 724 Feb 54

There are more than twenty appreciably different deoxyribonucleoside triphosphate assays using DNA polymerase in the literature. Therefore, each aspect of this method has been critically evaluated, including the purity of the substrates and of DNA polymerase, the reaction conditions, product isolation, and the effect of cell extracts on the assay. The final method uses a phosphatase-free DNA polymerase preparation, 2'-dAMP to inhibit the 3' leads to 5' exonuclease of DNA polymerase I, and includes a correction of both the background incorporation and the sample incorporation for dilution of the specific activity of the radioactive precursor by the sample. The sensitivity, range, accuracy, and reproducibility are reported as well as values for the deoxyribonucleoside triphosphate content of Chinese hamster ovary K-1 cells.
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PMID:Determination of deoxyribonucleoside triphosphates using DNA polymerase: a critical evaluation. 731 18


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