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)

Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.
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PMID:Molecular biological characterization of the human foamy virus reverse transcriptase and ribonuclease H domains. 748 84

Previous studies showed that an isolated human immunodeficiency virus type 1 (HIV-1) RNase H domain expressed as a fusion protein is highly active in Mn2+, but activity was dependent on a hexahistidine tag located at either the carboxyl or amino terminus of the fusion protein (J. Smith and M. Roth, J. Virol. 67:4037-4049, 1993). It was postulated that a histidine tag can somehow provide a function normally associated with the DNA polymerase domain of HIV-1 reverse transcriptase. To determine the contributions of the DNA polymerase subdomains of HIV-1 reverse transcriptase to its RNase H activity, we have characterized the activity of isolated RNase H domains which include either portions of the connection, the entire connection, or both the thumb and connection as N-terminal extensions. Including increasing lengths of these domains at the N terminus of the RNase H resulted in a progressive increase in Mn(2+)-dependent RNase H activity that was independent of a histidine tag. Activity of the isolated RNase H domains was also stimulated by the addition of independently purified polymerase subdomains. Further, this stimulation was shown to be a result of direct physical interactions between the thumb, connection, and RNase H domains. The connection and thumb subdomains were shown to contribute to substrate binding. The fingers and palm subdomains were found to be essential for Mg(2+)-dependent RNase H activity.
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PMID:Contributions of DNA polymerase subdomains to the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase. 752 94

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
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PMID:Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus. 754 60

A previously described large Vermont kindred possessing a high incidence of venous thromboembolism with associated Type I protein C deficiency (1) has been genetically analyzed. All nine exons of the protein C gene, including both coding and non-coding regions, have been amplified from blood cell genomic DNA using the Tag DNA polymerase chain reaction (PCR) and primers corresponding to flanking intronic regions, and the products directly sequenced. An initial mutation (C-->T) resulting in Thr298-->Met was observed in one arm of the family exhibiting a history of thrombosis and protein C deficiency and was designated protein CVERMONT IIa. However, examination of the kindred member parent (male) of this arm and members of other arms of the kindred demonstrated that the mutation entered the arm via the genetically unrelated spouse. Further analysis of the father and members of other arms of the kindred revealed a different mutation (C insertion: CAT-->CCAT), resulting in a frameshift beginning at amino acid #107 (His-->Pro) and truncation of the protein at codon #119 of the mature protein. This mutation, called protein CVERMONT IIb, is associated with protein C deficiency and thrombosis throughout the kindred.
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PMID:Genetic analysis of a large kindred exhibiting type I protein C deficiency and associated thrombosis. 791 73

All known family B DNA polymerases contain a conserved region of amino acids, KX6-7YG, which appears to be correspond to the 'finger' alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. Toward the goal of establishing the evolutionary relationship between the family A and B DNA polymerases, we have employed site-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase. We have replaced the lysine-340 with three amino acids: histidine, asparagine and glutamic acid, respectively. Mutant DNA polymerases were overexpressed and purified to near homogeneity. Our results showed that the modification of the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. These results support the proposal that the KX6-7YG motif of the family B DNA polymerases may be analogous to the KX7YG motif of the family A DNA polymerases, suggesting that two family DNA polymerases share a common ancestor.
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PMID:Mutagenesis of a highly conserved lysine 340 of the PRD1 DNA polymerase. 791 20

Crystal structures of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli complexed with deoxynucleoside triphosphate (dNTP) or with pyrophosphate (PPi) determined to 3.9-A resolution by X-ray crystallography show these molecules binding within the cleft of the polymerase domain and surrounded by residues previously implicated in dNTP binding. The dNTP binds adjacent to the O-helix [Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., & Steitz, T. A. (1985a) Nature 313, 762-766] with its triphosphate moiety anchored by three positively charged residues, Arg 754, Arg 682, and Lys 758, plus His 734 and Gln 708. The dNTP binding site observed in the crystal is consistent with the results of chemical modification including cross-linking and is also near many of the amino acid residues whose mutation affects catalysis [Polesky, A. H., Steitz, T. A., Grindley, N. D. F., & Joyce, C. M. (1990) J. Biol. Chem. 265, 14579-14591; Polesky, A. H., Dahlberg, M. E., Benkovic, S. J., Grindley, N. D. F., & Joyce, C. M. (1992) J. Biol. Chem. 267, 8417-8428]. However, we conclude that the position of at least the dNMP moiety of dNTP in the binary complex is not likely to be the same as in its catalytically relevant complex with primer-template DNA.
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PMID:Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. 826 Apr 91

Mutation in the REC1 gene of Ustilago maydis is known to lead to a complex phenotype with alterations in DNA repair, recombination, mutagenesis, meiosis, and cell division. The predicted product of the REC1 gene is a polypeptide of 522 amino acid residues with a molecular mass of 56,866 daltons, with no overall sequence homology to any other known protein. The open reading frame of the REC1 gene placed by itself in a U. maydis expression vector was found to be sufficient to complement the rec1 mutant. Overexpression of REC1 in Escherichia coli gave rise to the anticipated 57-kDa product together with a 3'-->5' exonuclease activity. This activity was only present in cells overexpressing REC1 and its characteristics were distinguishable from the major bacterial nucleases, but it had certain enzymatic features in common with epsilon, the proofreading exonuclease subunit of E. coli DNA polymerase III holoenzyme. To facilitate isolation of the protein product from bacteria, the REC1 gene was overexpressed from a vector that fused a hexa-histidine-leader sequence onto the amino terminus, enabling the isolation of the HisREC1 product on an immobilized metal ion affinity column. The His-REC1 protein co-eluted with the novel exonuclease activity. Alignment of the amino acid sequence of the REC1 gene product with the conserved proofreading exonuclease motifs of DNA polymerases indicated significant homology.
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PMID:The REC1 gene of Ustilago maydis involved in the cellular response to DNA damage encodes an exonuclease. 827 78

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.
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PMID:Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta. 864 5

Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201. These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators. rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C). Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons. The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H. In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability. The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals. Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes. We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation. Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites.
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PMID:Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex. 866 50

The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.
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PMID:Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis. 901 52


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