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 sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase beta was investigated by individually changing each amino acid in this region by site-directed mutagenesis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their Km's for primer DNA were about 10-fold higher than that of the wild type, while Km's for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase beta with primer.
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PMID:Aspartic acid residues at positions 190 and 192 of rat DNA polymerase beta are involved in primer binding. 203 95

We have demonstrated that carcinogen damage to DNA induces the production of cellular factors that act in trans to enhance the asynchronous replication of polyoma viral DNA. Exposure of a polyoma virus-transformed rat cell line to benzo[a]pyrene-7,8-diol-9,10-oxide (BPDE), the ultimate carcinogenic metabolite of benzo[a]pyrene, led to the accumulation of heterogeneously sized free viral DNA molecules which contain polyoma origin sequences as well as cellular sequences that flank the integrated viral DNA. When the sequence gpt was linked to the polyoma early region and transfected into rat cells, it underwent asynchronous replication in response either to direct treatment of the transfected cells with BPDE, or to fusion of untreated transfected cells with normal cells previously exposed to BPDE. Transient arrest of the cell cycle by hydroxyurea, isoleucine deprivation or methotrexate caused a slight enhancement of viral DNA replication when compared with BPDE. Both aphidicolin, an inhibitor of DNA polymerase alpha, and 3-aminobenzamide, an inhibitor of poly[ADP]ribosyl transferase, caused marked inhibition of BPDE-induced viral DNA synthesis. The induction of a trans-acting factor in response to damage of cellular DNA may be relevant to synergistic interactions between environmental chemicals and DNA viruses in cell transformation and to the general phenomenon of gene amplification.
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PMID:Carcinogen induced asynchronous replication of polyoma DNA is mediated by a trans-acting factor. 301 4

Mouse fibroblasts arrested in G0 by isoleucine deprivation were inoculated with the autonomous parvovirus minute virus of mice (MVM). Infected cells were released from the G0 block by transfer to complete medium and their progression to and and through the S phase was monitored. The onset of viral and cellular DNA synthesis coincided, suggesting that cellular factor(s) required for MVM DNA replication became available as soon as cells entered the S phase. Cellular DNA synthesis was reduced to about 60% by MVM infection. However, this inhibition did not decrease significantly the overall rate of DNA replication in infected cells because it was compensated by concomitant viral DNA synthesis. MVM infection delayed the movement of the cells out of S phase by at least 5 h. At any time post-infection, more than 95% of both viral and cellular DNA synthesis was sensitive to inhibition by aphidicolin. Since this drug is highly specific for cellular DNA polymerase alpha, the data are consistent with a major role of this enzyme in the in vivo DNA replication of autonomous parvovirus. The assembly of 95% of virus progeny particles was concomitant with a late phase or viral DNA replication which accounted for 30% of the total viral DNA synthesized. The inhibition of this residual viral DNA replication by aphidicolin reduced dramatically the size of the burst of infectious particles; this observation concurs with other evidence to suggest that encapsidation is driven by a late replication event sensitive to this drug.
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PMID:Interrelation between viral and cellular DNA synthesis in mouse cells infected with the parvovirus minute virus of mice. 641 61

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes: nucleoside analogs and nonnucleoside inhibitors. The nonnucleoside inhibitors are quite specific for the polymerase activity of HIV-1 RT; they do not affect the polymerase activity of HIV-2 RT or the ribonuclease H (RNase H) activity of either HIV-1 RT or HIV-2 RT. Structural, biochemical, and genetic analyses showed that this group of inhibitors binds in a hydrophobic pocket near the polymerase active site. Mutations in amino acids that line this hydrophobic pocket, for example at tyrosine 181, tyrosine 188, or lysine 103, lead to enzymes that are resistant to the nonnucleoside inhibitors. We have investigated the enzymatic properties of two mutants of HIV-1 RT in which residues 181 and 188 were replaced by the corresponding amino acids in HIV-2 RT (tyrosine 181-->isoleucine and tyrosine 188-->leucine). The two tyrosine mutants closely resemble the wild-type HIV-1 RT in almost all the catalytic functions tested, including the heat stability, sensitivity of the DNA polymerase activity to inhibition by deoxynucleoside analogs, inhibition by the zinc chelator o-phenanthroline, and the Km values calculated for the DNA polymerase activity. There is, however, a slight difference in the effect of orthophenanthroline on the RNase H activity. In addition, there is a subtle disparity in the fidelity of DNA synthesis (analyzed by a mispair extension assay), thus indicating that these mutant RTs are not likely to confer any selective advantages or disadvantages to the variant virions over wild-type virus.
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PMID:Enzymatic properties of two mutants of reverse transcriptase of human immunodeficiency virus type 1 (tyrosine 181-->isoleucine and tyrosine 188-->leucine), resistant to nonnucleoside inhibitors. 752 32

Genetic and molecular analysis in Drosophila melanogaster identifies eight suppressor mutations in the second largest subunit of RNA polymerase II. The suppressor mutations fall into two classes: five are strong, result from the same serine to cysteine amino acid residue substitution and rescue one conditional lethal allele in the largest subunit of RNA polymerase II; three are mild, result from a change in the same methionine residue to either isoleucine or valine, are located seven amino acid residues away from the strong suppressors and rescue two conditional lethal alleles in the largest subunit. Sequence analysis of the three regions around these mutations demonstrates that they are located within highly conserved domains but fails to explain the observed genetic interactions. One of the conditional lethal alleles maps within a region previously reported to share sequence similarity to Escherichia coli DNA polymerase I. As the gross structure of RNA polymerase II and DNA polymerase I is similar, even though their primary sequence is not, we predict that more similarities exist but may be too highly divergent to be detected by normal homology searches. We identify the most similar regions between each of the three conserved domains of RNA polymerase II, identified as functionally important because of the mutations we isolated, and DNA polymerase I. Molecular modeling these regions of RNA polymerase II onto the tertiary structure of DNA polymerase I predicts that all lie adjacent to the DNA binding cleft in positions such that they could interact with the phosphate backbone of DNA. This juxtaposition of mutations in the two largest subunits of RNA polymerase II suggest a mechanism for their genetic interactions.
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PMID:Molecular modeling of RNA polymerase II mutations onto DNA polymerase I. 796 18

The same point mutation in the human cytomegalovirus UL97 open reading frame was found in three independently isolated ganciclovir-resistant mutants of strain AD169. Point mutations in the DNA polymerase genes of these strains have been previously identified (N.S. Lurain, K.D. Thompson, E.W. Holmes, and G.S. Read, J. Virol. 66:7146-7152, 1992). All three strains are, therefore, double mutants. To determine the contribution of the UL97 mutation to the high ganciclovir resistance of these mutants, the mutation from the ganciclovir-resistant strain D6/3/1 was transferred to the wild-type strain AD169 to produce the recombinant R6HS. The ganciclovir resistance of R6HS is 4-fold lower than that of D6/3/1 but 10-fold higher than that of AD169. R6HS, like AD169, is sensitive to the nucleotide analogs (S)-1-[(3-hydroxy-2-phosphonylmethoxy) propyl]adenine and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine. Ganciclovir phosphorylation in R6HS-infected cells was at the same reduced level as that found in cells infected with the parental mutant D6/3/1. The same G-to-T transversion at nucleotide 1380 in the UL97 coding sequence is present in both R6HS and D6/3/1. This mutation results in the substitution of isoleucine for methionine at amino acid residue 460. In an alignment of the R6HS UL97 amino acid sequence with the amino acid sequences of a wide range of protein kinase family members, methionine 460 lies within a highly conserved region which may function in nucleotide binding and phosphate transfer.
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PMID:Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. 820 15

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

A lysine residue, contained in the motif "Kx2h", has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of phi29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type phi29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx2h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3'-5' exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of phi29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of phi29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3'-5' exonuclease active site.
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PMID:An invariant lysine residue is involved in catalysis at the 3'-5' exonuclease active site of eukaryotic-type DNA polymerases. 923 1

We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo. The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro. To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants. Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine. Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine. The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513. The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity. The possible roles of RNA polymerase in control of ColE1 copy number are discussed.
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PMID:Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli. 988 6

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.
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PMID:The hydrophobic hinge region of rat DNA polymerase beta is critical for substrate binding pocket geometry. 1590 25


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