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 photoaffinity compound 8-azido-dATP was used as a probe for the deoxyribonucleoside triphosphate-binding site of the large fragment of DNA polymerase I. Azido-dATP specifically modified a saturable binding site within the Klenow fragment, and each of the four natural deoxyribonucleoside triphosphate substrates competed with labeling at this site in proportion to its binding constant, as previously defined by equilibrium dialysis. Analysis of tryptic peptides after azido-dATP modification revealed five major cross-linking products, which apparently arose from five distinct photoadducts formed near Tyr-766.
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PMID:Photoaffinity labeling of the Klenow fragment with 8-azido-dATP. 218 Sep 51

Both Escherichia coli DNA polymerase I (pol I) and the large fragment of pol I (Klenow) were found to bypass a site-specific cis-syn thymine dimer, in vitro, under standard conditions. A template was constructed by ligating d(pCGTAT[c,s]TATGC), synthesized via a cis-syn thymine dimer phosphoramidite building block, to a 12-mer and 19-mer. The site and integrity of the dimer were verified by use of T4 denV endonuclease V. Extension of a 15-mer on the dimer-containing template by either pol I or Klenow led to dNTP and polymerase concentration dependent formation of termination and bypass products. At approximately 0.15 unit/microL and 1-10 microM in each dNTP, termination one prior to the 3'-T of the dimer predominated. At 100 microM in each dNTP termination opposite the 3'-T of the dimer predominated and bypass occurred. Bypass at 100 microM in each dNTP depended on polymerase concentration, reaching a maximum of 20% in 1 h at approximately 0.2 unit/microL, underscoring the importance of polymerase binding affinity for damaged primer-templates on bypass. Seven percent bypass in 1 h occurred under conditions of 100:10 microM dATP:dNTP bias, 1% under dTTP bias, and an undetectable amount under either dGTP or dCTP bias. At 100 microM in each dNTP, the ratio of pdA:pdG:pdC:pdT terminating opposite the 3'-T of the dimer was estimated to be 37:25:10:28. Sequencing of the bypass product produced under these conditions demonstrated that greater than 95% pdA was incorporated opposite both Ts of the dimer and that little or no frame shifting took place.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:cis-syn thymine dimers are not absolute blocks to replication by DNA polymerase I of Escherichia coli in vitro. 218 42

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.
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PMID:NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment. 226 37

The cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of high-dose ara-C clinical protocols. In this study, the incorporation of [3H]-dThd and [3H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [3H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [3H]-dThd incorporation, DNA synthesis was not completely inhibited since [3H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [3H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [3H]-dThd to [3H]-dTTP. Within 30 min exposure to 10 microM ara-C, the rate of cellular [3H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [3H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [3H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5'-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [3H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [3H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 microM ara-C (60 microM and 200 microM, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology. 231 Nov 69

Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase.
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PMID:[Template-primer-dependent inactivation of DNA polymerase alpha from human placenta by 2',3'-epoxyadenosine-5'-triphosphate]. 234 86

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.
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PMID:Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV. 239 41

We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA). Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10. A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions. In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J. (1988) J. Biol. Chem. 263, 6068). Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation. The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide. Arginine 682 was found to be the target of FSBdA reactivity. We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.
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PMID:Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I. Identification of arginine 682 as the binding site and its implication in catalysis. 240 60

The yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates (Singh, H., and Dumas, L. B. (1984) J. Biol. Chem. 259, 7936-7940). In the presence of ATP substrate and poly(dT) template, the enzyme preparation synthesizes discrete-length oligoribonucleotides (apparent length 8-12) and multiples thereof. The unit length primers are the products of de novo processive synthesis and are precursors to the synthesis of the multimers. Multimeric length oligoribonucleotides are not generated by continuous processive extension of the de novo synthesis products, however, nor do they arise by ligation of unit length oligomers. Instead, dissociation and rebinding of a factor, possibly the DNA primase, results in processive extension of the RNA synthesis products by an additional modal length. Thus, catalysis by the yeast DNA primase can be viewed as repeated cycles of processive unit length RNA chain extension. Inclusion of dATP substrate results in three distinct transitions: (i) coupling of RNA priming to DNA synthesis, (ii) suppression of multimer RNA synthesis, and (iii) attenuation of primer length. The less than unit length RNA primers appear to result from premature DNA chain extension, not degradation from either end of the unit length primer. We discuss possible roles of DNA polymerase and DNA primase in RNA primer attenuation.
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PMID:Yeast DNA primase and DNA polymerase activities. An analysis of RNA priming and its coupling to DNA synthesis. 242 99

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

Effects of ATP and some other nucleotides (AMP, ADP, CTP, GTP, UTP and dATP) on reparative DNA synthesis and repair patch ligation in bleomycin-pretreated permeable mouse sarcoma cells were studied. Reparative DNA synthesis was significantly stimulated by 2.5 mM ATP, ADP or dATP. The stimulation was observed on both DNA polymerase alpha- and beta-dependent reparative DNA synthesis. ATP concentration required for repair patch ligation was much lower than that required for the stimulation of reparative DNA synthesis. An apparent Km value for ATP of the repair patch ligation was about 40 microM. ADP supported repair patch ligation after being converted into ATP by adenylate kinase in permeable cells.
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PMID:Effects of ATP and other nucleotides on DNA repair synthesis in bleomycin-pretreated permeable mouse sarcoma cells. 244 62


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