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)

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with 4-(oxoacetyl)-phenoxyacetic acid (OAPA) results in the loss of DNA polymerase as well as template-primer binding activity but has no effect on the RT-associated RNase-H activity. Binding stoichiometry revealed that approximately 3 mol of OAPA bound per mole of enzyme, when complete enzyme activation occurred. However, in the presence of template-primer, OAPA does not abolish polymerase activity and 2 mol of OAPA remains bound to 1 mol of enzyme. This observation suggests that only one OAPA reactive site is responsible for the loss of polymerase activity. This site was located on a single tryptic peptide by comparing the maps of the native enzyme and the enzyme treated with OAPA in the presence and absence of template-primer. The appearance of a new peptide peak eluting at 125 min from a C-18 reverse-phase column was consistently noted in the tryptic digest of enzyme treated with OAPA. This peak was absent in tryptic peptides made from the control enzyme or the enzyme protein that was treated with OAPA in the presence of activated DNA or synthetic template-primers. Amino acid composition and sequence analyses of this peptide revealed that it spanned residues 312-342 in the primary amino acid sequence of MuLV RT. Since this peptide does not contain arginine residues and Lys-329 exhibited resistance to tryptic digestion, we conclude that Lys-329 is the target of OAPA action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysine-329 of murine leukemia virus reverse transcriptase: possible involvement in the template-primer binding function. 169 96

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.
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PMID:Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction. 184 97

Primer protein (PP) of bacteriophages M2 and phi 29 contains an Arg-Gly-Asp (RGD) sequence. The RGD-mediated protein-protein interaction in protein-primed DNA replication of M2 was studied in vitro using three purified and indispensable components: PP, DNA polymerase (POL) and template DNA linked to terminal protein (TP). PP competed with a synthetic RGD peptide for binding to the template DNA-TP complex (TP-DNA). In addition, POL bound to template TP-DNA only when complexed with PP. These results indicate that the RGD sequence of PP is responsible for the interaction of the PP-POL complex with TP-DNA, which contains the initiation site for the protein priming of DNA synthesis. At the moment when PP converts to TP upon linking the first deoxynucleotide, a conformational change results in exposure of the RGD binding site.
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PMID:Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide. 203 31

By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction. Replacement of Phe-181 by alanine reduced the enzyme activity only 30%. Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively. The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate. Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold. Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta.
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PMID:Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition. 219 36

A group of proteins that act as primers for initiation of linear DNA replication are called DNA-terminal proteins (terminal proteins). We have found a short stretch of conserved amino acid sequence among the terminal proteins from six different sources. The location of this sequence motif is also similar among the different terminal-proteins. To determine the functional role of this terminal-protein domain in DNA replication, we have studied the bacteriophage PRD1 system. The PRD1 terminal protein and DNA polymerase genes were cloned into expression vectors, and the recombinant plasmids were used for constructing PRD1 terminal protein mutants. Site-directed mutagenesis and functional analysis showed that one of the two arginines (Arg-174) in the conserved sequence is critical for the initiation complex-forming activity of the PRD1 terminal protein. Replacement of Arg-174 by noncharged amino acids resulted in nonfunctional terminal protein. Phenylglyoxal, an alpha-dicarbonyl compound that reacts with the guanidino group of arginine, inhibits initiation complex formation between PRD1 terminal protein and dGMP. On the basis of these results, we propose that Arg-174 represents, at least in part, the binding site for phosphate groups of dGTP.
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PMID:An essential arginine residue for initiation of protein-primed DNA replication. 223 78

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. The unusually high number of serine, leucine, and arginine residues in secretin has precluded the use of oligonucleotides to screen cDNA libraries to isolate a secretin cDNA. In the present study, a short cDNA encoding porcine secretin was amplified from duodenal mucosal first-strand cDNA template by using 16,384- and 4096-fold degenerate primers in the DNA polymerase chain reaction. From the sequence of the amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a cDNA library. Here we report the sequences of cDNAs encoding the porcine and rat secretin precursors. The predicted amino acid sequences reveal that each precursor consists of a signal peptide, an N-terminal peptide, secretin, and a 72-amino acid C-terminal peptide. Secretin has been highly conserved through evolution. Rat secretin differs from its porcine counterpart by a single glutamine-for-arginine substitution at position 14. In contrast, the amino acid sequences of the C-terminal peptides are only 39% conserved between the two species, suggesting that the C-terminal peptide does not have an essential physiologic function. RNA blot hybridizations reveal that the rat secretin gene is expressed throughout the small intestine. Although secretin immunoreactivity has been localized in the central nervous system by some laboratories, we are unable to detect secretin mRNA in tissues of the central nervous system by Northern blot hybridization.
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PMID:Secretin: structure of the precursor and tissue distribution of the mRNA. 231 22

We have labeled a template primer-dependent substrate deoxynucleoside triphosphate binding domain in Escherichia coli DNA polymerase I using an affinity labeling analogue of dATP, the 5'-fluorosulfonylbenzoyldeoxyadenosine (FSBdA). Using enzyme-template primer complex as a test system, we find that FSBdA-mediated inactivation occurs only when the template in the enzyme-template primer complex is poly(dT).(dA)10. A ribonucleotide analogue, 5'-fluorosulfonylbenzoyladenosine (FSBA) is not an effective inactivator under these conditions. In the absence of template primer, however, deoxyribonanalogue (FSBdA) irreversibly inactivates polymerase activity with characteristics similar to those reported for FSBA (Pandey, V.N., and Modak, M.J. (1988) J. Biol. Chem. 263, 6068). Binding stoichiometric studies in the presence and absence of template primer revealed that only 1 mol of FSBdA is incorporated per mol of enzyme which results in complete inactivation. The site of FSBdA action was investigated by comparative tryptic peptide mapping, followed by amino acid composition analysis of the modified peptide. Arginine 682 was found to be the target of FSBdA reactivity. We therefore conclude that the domain containing Arg-682 plays a major role in template-dependent dNTP binding and polymerization.
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PMID:Template primer-dependent binding of 5'-fluorosulfonyl-benzoyldeoxyadenosine by Escherichia coli DNA polymerase I. Identification of arginine 682 as the binding site and its implication in catalysis. 240 60

The incorporation of labeled precursors into DNA, RNA and protein in phytohaemagglutinin (PHA)-prestimulated human lymphocytes was maximally inhibited by liver extract (LEx) or arginase at 24 h. The activities of DNA polymerase alpha, beta and gamma were less inhibitable by these agents than was [3H]thymidine incorporation. The inhibition of DNA, RNA and protein syntheses by either LEx or arginase is probably due to arginine depletion by arginase activity, since their syntheses were similarly inhibited when cultured in an arginine-free medium in the absence of arginase. These results indicate that arginase nonspecifically inhibits the activities of DNA polymerase. The inhibition is probably due to arginine depletion.
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PMID:The mechanisms of the inhibitory effects of liver extract on lymphocyte proliferation. III. The effects of arginase on DNA polymerase activities. 247 98

The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages phi 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of protein-primed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of phi 29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.
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PMID:An inhibitory effect of RGD peptide on protein-priming reaction of bacteriophages phi 29 and M2. 260 28


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