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 deoxythymidine-5'-triphosphate (dTTP) analog 3'-fluorothymidine 5'-triphosphate (3'-FdTTP) inhibits DNA synthesis by T4 wild-type, L98 (mutator) and CB121 (antimutator) DNA polymerase. CB121 DNA polymerase is less sensitive by a factor of two than the L98 and T4+ enzymes. Inhibition is not due to incorporation of the analog into DNA. 3'-FdTTP acts competitively to the substrate dTTP. The CB121 polymerase exhibits a higher Ki to Km ratio than the other two enzymes (5.3 vs. 3.3) and thus discriminates better between the substrate dTTP and its analog 3'-FdTTP. 3'-FdTTP inhibits the polymerase-associated 3'-5' exonuclease activities to the same extent as their polymerase activities. The CB121 3'-5' exonuclease activity is suppressed only half as much by 3'-FdTTP as by dTTP. The results are discussed in relation to the role of T4 DNA polymerase and its associated 3'-5' exonuclease in determining the accuracy of DNA replication.
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PMID:Inhibitor studies on phage T4 wild-type and mutant DNA polymerase. IV. The substrate analog 3'-fluorothymidine 5'-triphosphate. 722 44

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.
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PMID:Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus. 746 44

The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.
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PMID:The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure. 748 14

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.
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PMID:The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma. 757 60

The polA gene (encoding DNA polymerase I) from Mycobacterium tuberculosis was cloned using an internal gene segment probe generated by PCR amplification of genomic DNA [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide 904 amino acids (aa) in length that shares 89% identity with a 911-aa homologue from Mycobacterium leprae. The polypeptide has all of the primary structural elements necessary for DNA polymerase and 5'-3' exonuclease activity, but lacks the motifs required for an associated 3'-5' exonuclease (proofreading) activity.
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PMID:Cloning and sequence analysis of the gene encoding the DNA polymerase I from Mycobacterium tuberculosis. 759 Mar 2

Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis. Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (ErR) mt mutants in yeast mapped to the 3'-5' exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting the importance of proofreading in accurate mt DNA replication. The essential role of the invariant glutamate at the Exo1 site was confirmed and the participation of four amino acids (aa) in the 3'-5' Exo function revealed. Another mutation that is located between the Exo1 and Exo2 sites produced an extremely strong mutator phenotype associated with impaired DNA replication, but could be assigned neither to a conserved aa nor to a conserved portion of the 3'-5' exonuclease domain. The importance of the polymerization domain in accurate mt DNA replication was pointed out by three mutator mutations. Two of these severely impaired mt DNA replication and were assigned to a subdomain of the polymerase which probably corresponds to the 'fingers' module of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk). The third, which did not alter the efficiency of DNA replication, was located at the active center of the polymerization reaction. Finally, the mutation, R1001I, mapped to the C-terminal part of the MIP1 protein which has no counterpart in prokaryotic DNA polymerases.
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PMID:Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-encoding MIP1 gene from Saccharomyces cerevisiae. 762 2

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).
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PMID:Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin. 763 Jul 15

The DNA polymerase from Thermus aquaticus (Taq polymerase), famous for its use in the polymerase chain reaction, is homologous to Escherichia coli DNA polymerase I (pol I) Like pol I, Taq polymerase has a domain at its amino terminus (residues 1-290) that has 5' nuclease activity and a domain at its carboxy terminus that catalyses the polymerase reaction. Unlike pol I, the intervening domain in Taq polymerase has lost the editing 3'-5' exonuclease activity. Although the structure of the Klenow fragment of pol I has been known for ten years, that of the intact pol I has proved more elusive. The structure of Taq polymerase determined here at 2.4 A resolution shows that the structures of the polymerase domains of the thermostable enzyme and of the Klenow fragment are nearly identical, whereas the catalytically critical carboxylate residues that bind two metal ions are missing from the remnants of the 3'-5' exonuclease active site of Taq polymerase. The first view of the 5' nuclease domain, responsible for excising the Okazaki RNA in lagging-strand DNA replication, shows a cluster of conserved divalent metal-ion-binding carboxylates at the bottom of a cleft. The location of this 5'-nuclease active site some 70 A from the polymerase active site in this crystal form highlights the unanswered question of how this domain works in concert with the polymerase domain to produce a duplex DNA product that contains only a nick.
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PMID:Crystal structure of Thermus aquaticus DNA polymerase. 763 14

DNA sequence analysis revealed a gene encoding the Marek's disease virus (MDV) DNA polymerase (pol) within the BamHI-E fragment of the long unique region of the virus genome. Identification is based on an extensive amino acid homology between the MDV open reading frame and the DNA pol (UL30) of the herpes simplex virus. We describe here a 3540-base-pair fragment of the MDV DNA encoding 1180 amino acids with a M(r) of 133,920 daltons as the viral DNA pol gene, with the analysis of transcription and translation. In Northern blot hybridization, a transcript of 4.0 kb was detected in GA-MDV-infected duck embryo fibroblast (DEF) cells. An antiserum was generated in rabbit using TryE-pol fusion protein expressed in E. coli. This antiserum specifically immunoprecipitated a protein of 135 kD from lysates of MDV-GA-infected DEF cells. MDV DNA pol showed extensive homology to five distantly related herpesviruses: equine herpesvirus (EHV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV). Comparison of amino acid sequences among the herpesviruses highlights nine highly conserved regions. Three of the conserved regions are in the N-terminus in the 3'-5' exonuclease domains and the remaining six are in the C-terminus in the catalytic domains. The predicted structural characters are in good agreement with the published data on a number of human herpesvirus DNA pol. The identification of MDV DNA pol gene may lead to a better understanding of MDV replication.
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PMID:Identification and characterization of a Marek's disease virus gene encoding DNA polymerase. 765 4

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.
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PMID:Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication. 766 51


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