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 genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.
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PMID:Fidelity of HIV-1 reverse transcriptase copying RNA in vitro. 137 Sep 10

5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C-->T transitions, resulting from spontaneous deamination of mC to T. The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesis via G-->A transitions. Stimulation of the G-->A mutational pathway could involve preferential misincorporation of dAMP opposite template mC compared to C. To investigate this possibility, we synthesized a sequence containing mC at a defined template location. We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity. Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase. AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite mC than C. Favored extension of a 5-MeC.A over C.A mispair was also observed with a difference of about 3-fold. In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either mC or C sites or when extending mispairs involving mC and C. Incorporation of dAMP opposite either C or mC was barely detectable using pol alpha, although pol alpha has been observed to form A.C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-->A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.
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PMID:A comparison of the fidelity of copying 5-methylcytosine and cytosine at a defined DNA template site. 138 39

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.
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PMID:Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication. 140 68

The incorporation of cytosine arabinoside monophosphate (araCMP) into DNA at internucleotide linkages by DNA polymerase alpha (DNA pol alpha) has been investigated by using oligonucleotide primed DNA templates. The products of reactions catalyzed by DNA pol alpha in vitro were analyzed on polyacrylamide gels to measure insertion of araCMP, extension from an araCMP 3' terminus, and binding of the enzyme to an araCMP 3' terminus. The results show that insertion of araCMP opposite dGMP in the DNA template is about 3-fold less efficient than insertion of dCMP. Extension from an araCMP 3' terminus by addition of the next complementary nucleotide is approximately 2000-fold less efficient than extension from a correctly base-paired 3' terminus. In the absence of the second substrate, dNTP, DNA pol alpha binds with approximately equal affinities to DNA templates that contain oligonucleotide primers with araCMP or dCMP positioned at the 3' terminus. In the presence of dNTP, the enzyme extends the araCMP 3' terminus or dissociates, but it is not trapped at the araCMP 3' terminus in a nonproductive ternary complex as is observed at the ddCMP 3' terminus. To determine if slow phosphodiester bond formation contributes to the observed extension rate from the araCMP 3' terminus by DNA pol alpha, oligonucleotide primers with araCMP positioned at the 3' terminus were elongated by addition of the alpha-phosphorothioate analogue of the next complementary nucleotide. The rate of extension from araCMP by addition of 2'-deoxyadenosine 5'-O-phosphorothioate (dAMP alpha S) was 6-fold slower than by addition of dAMP, indicating that bond formation is partially rate limiting in the extension reaction. Thus, inefficient extension from the araCMP 3' terminus is the major determinant contributing to the low incorporation frequency of araCMP into DNA by DNA pol alpha, and this inefficiency can be attributed, in part, to slower phosphodiester bond formation at the araCMP 3' terminus.
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PMID:Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase alpha. 142 52

A mispair-specific 3'-->5' exonuclease copurifies quantitatively with the near-homogeneous Drosophila gamma polymerase (Kaguni, L.S., and Olson, M.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6469-6473). The exonuclease and polymerase exhibit similar reaction requirements and optima, suggesting functional coordination of their activities. Under nonpolymerization conditions, the 3'-->5' exonuclease hydrolyzes 3'-terminal mispairs approximately 15-fold more efficiently than 3'-terminal base pairs on primed single-stranded DNA substrates, whereas it does not discriminate between any of three specific mispairs (dAMP:dAMP;dGMP:dGMP; dGMP:dAMP). Under polymerization conditions, gamma polymerase does not extend a 3'-terminal mispair from the "stationary" state, even in the presence of a large excess of the next correct nucleotide. Instead, 3'-terminal mispairs are hydrolyzed quantitatively by the 3'-->5' exonuclease over the reaction time course. During DNA synthesis by gamma polymerase in the "polymerization" mode, limited misincorporation and subsequent mispair extension do occur. Here, it appears that misincorporation and not mispair extension is rate-limiting. Template-primer challenge experiments suggest that the mechanism of template-primer transfer from the 3'-->5' exonuclease active site to the DNA polymerase active site is intermolecular; transfer from the exonuclease to polymerase mode appears to require dissociation and reassociation of mitochondrial DNA polymerase.
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PMID:3'-->5' exonuclease in Drosophila mitochondrial DNA polymerase. Substrate specificity and functional coordination of nucleotide polymerization and mispair hydrolysis. 142 61

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. 170 52

In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.
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PMID:DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase. 173 Jun 46

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.
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PMID:In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents. 174 Dec 92

The kinetic parameters governing incorporation of correct and incorrect bases into synthetic DNA duplexes have been investigated for Escherichia coli DNA polymerase I [Klenow fragment (KF)] and for two mutants, Tyr766Ser and Tyr766Phe. Tyr766 is located at the C-terminus of helix O in the DNA-binding cleft of KF. The catalytic efficiency for correct incorporation of dNTP is reduced 5-fold for Tyr766Ser. The catalytic efficiencies of all 12 possible misincorporations have been determined for both KF and Tyr766Ser by using single-turnover kinetic conditions and a form of the enzyme that is devoid of the 3'-5' exonuclease activity because of other single amino acid replacements. Tyr766Ser displays an increased efficiency of misincorporation (a reduction in fidelity) for several of the 12 mismatches. The largest increase in efficiency of misincorporation for Tyr766Ser occurs for the misincorporation of TMP opposite template guanosine, a 44-fold increase. In contrast, the efficiencies of misincorporation of dAMP opposite template A, G, or C are little affected by the mutation. A determination of the kinetic parameters associated with a complete kinetic scheme has been made for Tyr766Ser. The rate of addition of the next correct nucleotide onto a preexisting mismatch is decreased for Tyr766Ser. The fidelity of Tyr766Phe was not substantially different from that of KF for the misincorporations examined, indicating that it is the loss of the phenolic ring of the side chain of Tyr766 that leads to the significant decrease in fidelity. The results indicate that KF actively participates in the reduction of misincorporations during the polymerization event and that Tyr766 plays an important role in maintaining the high fidelity of replication by KF.
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PMID:A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity. 189 34

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-). 199 Nov 25


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