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 (-) enantiomer of 3'-thiacytidine (lamivudine) has been found to be a potent inhibitor of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) replication. Mutation of methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HIV reverse transcriptase has been shown to be responsible for lamivudine resistance in HIV. The hepadnaviruses also have the YMDD motif in their DNA polymerase. Therefore, it is possible that hepadnaviruses could develop lamivudine resistance by a similar mutation at this motif. We analyzed the HBV from a liver transplantation patient who developed recurrent HBV viremia during lamivudine treatment. The polymerase gene was amplified by polymerase chain reaction (PCR), and the region coding for the YMDD motif was sequenced. The pretreatment HBV sequence coded for YMDD, while the lamivudine-resistant mutant HBV coded for YIDD (tyrosine, isoleucine, aspartate, aspartate). With the documented changes in the YMDD motif of lamivudine-resistant HIV, it is likely that the methionine-to-isoleucine mutation in the YMDD motif of the HBV polymerase contributes significantly to the lamivudine-resistance of HBV isolated from this patient.
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PMID:Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. 878 48

Famciclovir (FCV) and lamivudine (LAM) reduce viral replication in patients with recurrent hepatitis B virus (HBV) infection after orthotopic liver transplantation (OLT). Eighteen of 20 patients with insufficient response to FCV were treated with 100 mg LAM daily after OLT. These patients had shown nonresponse (n = 5), partial response (n = 7), or breakthrough (n = 6) during FCV therapy. Despite passive immunoprophylaxis with hepatitis B immunoglobulin after liver transplantation, HBV reinfection had occurred in 14 of 15 transplanted patients. HBV-DNA levels and the regions A to E of the HBV-DNA polymerase gene were analyzed before and after treatment failure to either therapy. Within 4 weeks on LAM, all but 1 patient showed a 95% average reduction of the HBV-DNA level. As with FCV, we did not observe any severe side-effects attributable to LAM. However, 7 patients developed a breakthrough within 12, 29 (n = 2), 32, 37, 54, and 145 weeks under treatment with LAM associated with the methionine-to-valine signature mutation (M552V) in the YMDD motif in all. With FCV, no unique, but a dominant, resistance pattern with the L528M mutation was identified for patients with breakthrough under FCV. In contrast, nonresponders or patients with partial response to FCV did not exhibit such mutations. Our results indicate that the L528M mutation is a risk factor for LAM breakthrough, because breakthrough during LAM occurred earlier in patients with this mutation (50 +/- 10 weeks vs. 120 +/- 21 weeks). Because breakthrough on either treatment is frequent for this specific group of patients, the use of combination therapy should be explored.
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PMID:Mutational pattern of hepatitis B virus on sequential therapy with famciclovir and lamivudine in patients with hepatitis B virus reinfection occurring under HBIg immunoglobulin after liver transplantation. 1038 63

Three conserved motifs (named A, B and C) have been proposed to form the polymerization active site in all classes of DNA-dependent polymerases. In eukaryotic-type (alpha-like) DNA polymerases, motif A is characterized by the consensus "Dx2SLYP". Mutants in phi29 DNA polymerase residue Tyr254 of this conserved motif had been previously shown to be affected in dNTP binding. Here, we show that a single substitution of Tyr254 into a valine residue enables the enzyme to incorporate ribonucleotide substrates, without affecting its wild-type affinity for dNTPs. Whereas the wild-type enzyme preferred dNTPs more than two million-fold over rNTPs, the mutation of Tyr254 into valine reduced the discrimination for rNTPs up to 1000-fold. In addition to this discrimination mechanism, based on sugar selection, phi29 DNA polymerase is very inefficient when extending an RNA primer terminus, allowing its exonucleolytic degradation. These results indicate that the Tyr254 of phi29 DNA polymerase is responsible for the discrimination against the 2'-OH group of an incoming ribonucleotide. This is the first time that the invariant tyrosine residue of motif A is involved in ribo- versus deoxyribonucleotide discrimination in an eukaryotic-type DNA polymerase.
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PMID:A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase. 1038 70

Structures of DNA polymerase (pol) beta bound to single-nucleotide gapped DNA had revealed that the lyase and pol domains form a "doughnut-shaped" structure altering the dNTP binding pocket in a fashion that is not observed when bound to non-gapped DNA. We have investigated dNTP binding to pol beta-DNA complexes employing steady-state and pre-steady-state kinetics. Although pol beta has a kinetic scheme similar to other DNA polymerases, polymerization by pol beta is limited by at least two partially rate-limiting steps: a conformational change after dNTP ground-state binding and product release. The equilibrium binding constant, K(d)((dNTP)), decreased and the insertion efficiency increased with a one-nucleotide gapped DNA substrate, as compared with non-gapped DNA. Valine substitution for Asp(276), which interacts with the base of the incoming nucleotide, increased the binding affinity for the incoming nucleotide indicating that the negative charge contributed by Asp(276) weakens binding and that an interaction between residue 276 with the incoming nucleotide occurs during ground-state binding. Since the interaction between Asp(276) and the nascent base pair is observed only in the "closed" conformation of pol beta, the increased free energy in ground-state binding for the mutant suggests that the subsequent rate-limiting conformational change is not the "open" to "closed" structural transition, but instead is triggered in the closed pol conformation.
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PMID:DNA structure and aspartate 276 influence nucleotide binding to human DNA polymerase beta. Implication for the identity of the rate-limiting conformational change. 1102 43

Mutated constituents of the DNA replication complex might contribute to the mutational load of the genome during tumor development by impairing DNA synthesis as well as cell cycle-related control of DNA replication. To prove or disprove this hypothesis, we looked for mutations in the cDNA sequences of the four subunits of DNA polymerase alpha-primase from both highly malignant Novikoff hepatoma cells and regenerating normal rat liver and compared physicochemical and catalytic properties of the DNA polymerase alpha-primase complexes purified from both sources. Sequence analysis showed two mutations in subunit B from Novikoff cells: one in nucleotide position 855 (CCG-->CCA) that did not result in an amino acid exchange and one in position 862 (GTG-->ATG) that caused a change of valine to methionine in codon 288. No mutation was found in the three other subunits. The wild-type and mutated sequences of subunit B were cloned and expressed in vitro. Sedimentation analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformation of subunit B. The analysis of the purified DNA polymerase alpha-primase complexes showed a sedimentation value that was significantly higher for the enzyme complex from normal liver than for that from Novikoff cells. In addition, DNA polymerase alpha-primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally related compounds (such as (R,S)-7-ethyl-10-hydroxy camptothecin, 9-aminocamptothecin, and 10-hydroxycamptothecin) than the enzyme from normal rat liver. Thus, the amino acid change found in subunit B appears to result in a conformational change of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells. Whether this mutation influences genetic instability or tumor development needs to be explored.
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PMID:A mutation in subunit B of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells concomitant with a conformational change and abnormal catalytic properties of the DNA polymerase alpha-primase complex. 1153 67

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.
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PMID:A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive. 1155 38

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.
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PMID:Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines, a novel class of herpesvirus antiviral agents. 1252 21

A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the alpha-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.
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PMID:A temperature-sensitive mutation in the dnaE gene of Caulobacter crescentus that prevents initiation of DNA replication but not ongoing elongation of DNA. 1476 18

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3'-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent K(m) values for dNTP. Discrimination between AZTTP (3'-azido-3'-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5-6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G-->A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.
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PMID:Nucleotide specificity of HIV-1 reverse transcriptases with amino acid substitutions affecting Ala-114. 1554 34

Moloney murine leukemia virus reverse transcriptase (RT) selectively uses deoxyribonucleotides over ribonucleotides (rNTPs) as substrates. Substitution of F155 with valine (F155V) was previously found to increase the enzyme's affinity for rNTPs, though without affecting the V(max) for catalysis, and thereby conferred to the enzyme significant RNA polymerase activity. We have sought new mutations that might increase the RNA polymerase activity of the F155V mutant. We report here that substitution of Q84 with alanine improved RT-F155V's RNA polymerase activity, but also its DNA polymerase activity. Kinetic analysis and gel-retardation assays suggested that the substitution increased the enzyme's general affinity for the template-primer.
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PMID:Gln(84) of moloney murine leukemia virus reverse transcriptase regulates the incorporation rates of ribonucleotides and deoxyribonucleotides. 1646 20


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