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)

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.
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PMID:A meiotic DNA polymerase from a mushroom, Agaricus bisporus. 817 91

A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.
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PMID:Detection and analysis of diverse herpesviral species by consensus primer PCR. 878 66

African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3'-5'-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase beta (pol beta), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol beta-type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.
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PMID:Characterization of an African swine fever virus 20-kDa DNA polymerase involved in DNA repair. 938 36

Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
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PMID:Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes. 968 90

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.
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PMID:Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR. 1059 86

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.
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PMID:Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain. 1066 12

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.
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PMID:DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA. 1259 53

Reverse transcriptases (RTs) are multidomain enzymes of variable architecture that couple both RNA- and DNA-directed DNA polymerase activities with an RNase H activity specific for an RNA:DNA hybrid in order to replicate the single-stranded RNA genome of the retrovirus. Previous structural work has been reported for the heterodimeric HIV-1 and HIV-2 RTs. We now report the first crystal structure of the full-length Moloney murine leukemia virus (MMLV) RT at 3.0 A resolution. The structure reveals a clamp-shaped molecule resulting from the relative positions of the thumb, connection, and RNase H domains that is strikingly different from the HIV-1 RT and provides the first example of a monomeric reverse transcriptase. A comparative analysis with related DNA polymerases suggests a unique trajectory for the template-primer exiting the polymerase active site and provides insights regarding processive DNA synthesis by MMLV RT.
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PMID:The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus. 1513 Apr 74

Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation.
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PMID:Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene. 1566 55

Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.
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PMID:Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair. 1922 23


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