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)

Three different subspecies of DNA polymerase alpha from calf thymus sedimenting at 9 S, 7 S and 5.7 S have been investigated with respect to their accuracy of in vitro DNA synthesis on poly (dA) (dT)16 and poly d(AT) as template-primers. Our results indicate that the structure of DNA polymerase alpha has a strong influence on the accuracy of DNA synthesis. The 9 S enzyme shows a misincorporation frequency of about 1:100 000. An error rate of 1:15 000 is measured for the 7 S species. The 5.7 S enzyme for which an error rate of 1:3 000 is determined, has to be considered as error prone. Lowering the rate of DNA synthesis leads to a decrease in fidelity. The single stranded DNA binding protein from E.coli increases the accuracy of the 5.7 S and the 7 S enzyme by a factor of two. Mn2+ decreases the fidelity of all three subspecies in a concentration dependent manner.
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PMID:Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers. 686 63

We have identified three lesions rather than cyclobutane dimers which alter the properties of UV-irradiated poly(dC) as a template for E.coli DNA polymerase I, and have characterised these lesions with respect to their coding properties, rates of formation and decay, and their sensitivity to uracil DNA glycosylase. Our results lead us to conclude that these lesions are (1) cytosine hydrates, which code for cytosine and to a lesser extent thymine, (2) uracil hydrates, which code for adenine and are not sensitive to uracil DNA glycosylase, and (3) uracils, which code for adenine and are removed by uracil DNA glycosylase.
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PMID:Mechanism of ultraviolet-induced mutagenesis: the coding properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I. 702 15

We have examined the mutagenic properties of the T-T pyrimidine (6-4) pyrimidinone UV photoproduct in Saccharomyces cerevisiae, transforming the yeast cells either with single-stranded vectors that carried this adduct at a unique site or with gapped duplex vectors in which the adduct was located within a 28 nt single-stranded region. In an earlier study with SOS-induced Escherichia coli, we found that this photoproduct is highly mutagenic, specifically generating 3' T-->C substitutions in >85% of replicated molecules, and ascribed this specificity to the formation of a stable guanine-pyrimidinone mispair via hydrogen bonds at N-3 and O-2. In contrast, this adduct is very much less mutagenic in yeast, with 60-70% of molecules being replicated accurately and only 12-20% of them exhibiting 3' T-->C substitutions. The enhanced accuracy may reflect the ability of a yeast DNA polymerase, but not E.coli DNA polymerase III, to trap the adduct in a configuration favorable for the formation of an adenine-pyrimidinone base pair.
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PMID:The T-T pyrimidine (6-4) pyrimidinone UV photoproduct is much less mutagenic in yeast than in Escherichia coli. 759 18

The ability of Escherichia coli DNA polymerase I to retrotranscribe an RNA template was examined under steady-state conditions, using a primer extension assay which allows determination of kinetic constants on well-defined heterogeneous sequences. Equilibrium and rate constants for the initial binding step of the enzyme to two homologous DNA and RNA templates do not show striking differences. In both cases, under steady-state conditions, processivity limits the maximal velocity of the translocation process. The lower catalytic efficiency of the enzyme when it operates on RNA is then reflected by a 100-fold greater apparent average Michaelis constant for the deoxynucleotide substrates. We conclude that E.coli DNA polymerase I effectively transcribes both templates, its performances being limited in both cases by its intrinsically low processivity. Furthermore, DNA polymerase I is a strikingly accurate enzyme when operating on RNA. Magnesium has to be substituted by manganese so that a pattern of errors could be detected. This great accuracy results from a combination of factors. The 3' to 5' exonuclease activity is still operating but in a non-discriminative manner. Elongation of a mismatched primer terminus is markedly impaired. The forward polymerization rate of incorporation of an incorrect deoxynucleotide must be extremely low, when Mg2+ is present. In summary E.coli DNA polymerase I preserves its main characteristics when retrotranscribing RNA.
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PMID:E. coli DNA polymerase I as a reverse transcriptase. 767 88

The heat-shock 70 protein (Hsp70) chaperone family is very conserved and its prokaryotic homologue, the DnaK protein, is assumed to form one of the cellular systems for the prevention and restoration of heat-induced protein denaturation. By using anti-DnaK antibodies we purified the DnaK homologue heat-shock cognate protein (Hsc70) from calf thymus to apparent homogeneity. This protein was classified as an eukaryotic Hsc70, since (i) monoclonal antibodies against eukaryotic Hsc70 recognized it, (ii) its amino-terminal sequence showed strong homology to Hsp70s from eukaryotes and, (iii) it had an intrinsic weak ATPase activity that was stimulated by various peptide substrates. We show that this calf thymus Hsc70 protein protected calf thymus DNA polymerases alpha and epsilon as well as Escherichia coli DNA polymerase III and RNA polymerase from heat inactivation and could reactivate these heat-inactivated enzymes in an ATP-hydrolysis dependent manner, likely leading to the dissociation of aggregates formed during heat inactivation. In contrast to this, DnaK protein was exclusively able to protect and to reactivate the enzymes from E.coli but not from eukaryotic cells. Finally, the addition of calf thymus DnaJ co-chaperone homologue reduced the amount of Hsc70 required for reactivation at least 10-fold.
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PMID:Calf thymus Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes. 779 40

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.
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PMID:Family A and family B DNA polymerases are structurally related: evolutionary implications. 781 3

DNA polymerase preferentially incorporate dAMP opposite abasic sites (A-rule). The mechanism of the A-rule can be studied by analyzing three dissected stages of the reaction including (i) initial nucleotide insertion, (ii) proofreading excision of the inserted nucleotide and (iii) extension of the nascent primer terminus. To assess the role of the stage (ii) in the A-rule, kinetic parameters of the proofreading excision of primer terminus nucleotides opposite abasic sites were determined using E.coli DNA polymerase I Klenow fragment. The relative efficiency of the excision (Vmax/Km) revealed that removal of A was the least favored of the four nucleotides, but the differences in the efficiencies between excision of A and the other nucleotides was less than 2-fold. In addition, in an attempt to reconcile kinetic data associated with the stage (i) or (ii), the differences in free energy changes (delta delta G degrees) for the formation of model template-primer termini containing XN pairs (X = abasic site, N = A, G, C or T) were determined by temperature dependent UV-melting measurements. The order of delta delta G degrees was XG > XA = XC > or = XT, with delta delta G degrees being 0.5 kcal/mol for the most stable XG and the least stable XT. Based on these data, the role of the stage (ii) and energetic aspects of the A-rule are discussed.
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PMID:On the mechanism of preferential incorporation of dAMP at abasic sites in translesional DNA synthesis. Role of proofreading activity of DNA polymerase and thermodynamic characterization of model template-primers containing an abasic site. 787 May 77

Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E. coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.
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PMID:Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E. coli and in vitro. 793 3

An investigation was undertaken to study DNA replication in cultured human HeLa cells and Escherichia coli in response to nickel chloride (NiCl2). Treatment with NiCl2 increased both the rate of DNA replication and total cell number in HeLa cells and E. coli in a time- and concentration-dependent manner. The maximum stimulation of thymidine uptake into DNA was observed with 0.125-0.25 mM NiCl2 for both cell types. In studies of DNA replication using a crude HeLa cellular extract, NiCl2 at concentrations below 0.125 mM also induced a stimulation over the background of MgCl2-dependent [3H]dTMP incorporation into activated calf thymus DNA. However, a similar stimulatory effect from NiCl2 was not observed with either purified HeLa DNA polymerase alpha or E.coli DNA polymerase I Klenow fragment. In the absence of Mg2+, the low response of either DNA polymerase alpha or Klenow fragment to stimulation by Ni2+ was thought to be enhanced by the presence of Ni(2+)-binding proteins presented in the crude HeLa cell extract.
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PMID:The stimulatory effect of nickel chloride on DNA replication in human HeLa cells and Escherichia coli. 820 62

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.
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PMID:Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme. 826 41


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