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)

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.
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PMID:Substrate properties of 25-nt parallel-stranded linear DNA duplexes. 255 23

Yeast DNA polymerases I and III have been well characterized physically, biochemically, genetically and immunologically. DNA polymerase II is present in very small amounts, and only partially purified preparations have been available for characterization, making comparison with DNA polymerases I and III difficult. Recently, we have shown that DNA polymerases II and III are genetically distinct (Sitney et al., 1989). In this work, we show that polymerase II is also genetically distinct from polymerase I, since polymerase II can be purified in equal amounts from wild-type and mutant strains completely lacking DNA polymerase I activity. Thus, yeast contains three major nuclear DNA polymerases. The core catalytic subunit of DNA polymerase II was purified to near homogeneity using a reconstitution assay. Two factors that stimulate the core polymerase were identified and used to monitor activity during purification and analysis. The predominant species of the most highly purified preparation of polymerase II is 132,000 Da. However, polymerase activity gels suggest that the 132,000-Da form of DNA polymerase II is probably an active proteolytic fragment derived from a 170,000-Da protein. The highly purified polymerase fractions contain a 3'----5'-exonuclease activity that purifies at a constant ratio with polymerase during the final two purification steps. However, DNA polymerase II does not copurify with a DNA primase activity.
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PMID:Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae. 264 4

The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.
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PMID:How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment). 265 95

The modification of Klenow fragment of DNA polymerase I E. coli was investigated by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d(pT)2pC[Pt2+(NH3)2OH](pT)7. The template binding site of the enzyme was modified by these reagents in the presence of NaF (5 mM), which inhibits selectively the 3'----5'-exonuclease activity of the enzyme and therefore prevents the reagent from degradation. NaCN destroyed covalent bonds between reagents and enzyme, restoring activity of the Klenow fragment. The affinity of different ligands (inorganic phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of Klenow fragment was estimated. Minimal ligands capable to bind with the template site were shown to be triethylphosphate (Kd 290 microM) and phosphate (Kd 26 microM). Ligand affinity increases by the factor 1.76 per an added (monomer unit from phosphate to d(pT) and then for oligonucleotides d(Tp)nT (n 1 to 19-20). At n greater than 19-20, the ligand affinity remained constant. The complete ethylation of phosphodiester groups lowers affinity of the oligothymidylates to the enzyme by approximately 10 times, and comparable decrease of Pt2+-oligonucleotide affinity to polymerase is caused by the absence of Mn2+-ions. The data obtained led to suggestion that one Me2+-dependent electrostatic contact of the template phosphodiester group with the enzyme takes place (delta G = -1.45...-1.75 kcal/mole). Formation of a hydrogen bond with the oxygen atom of P = O group of the same template phosphate is also assumed (delta G = -4.8...-4.9 kcal/mole). Other template internucleotide phosphates do not interact with the enzyme but the bases of oligonucleotides take part in hydrophobic interactions with the template binding site. Gibbs energy changes by -0.34 kcal/mole when the template is lengthened by one unit.
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PMID:[Klenow fragment of DNA-polymerase I from E. coli. III. The role of internucleotide phosphate groups of the matrix in its binding with the enzyme]. 266 77

Reaction of DNA synthesis catalyzed by DNA polymerase I KF in the presence of 2'-deoxynucleoside 5'-alpha-thiotriphosphates (dNTP alpha S) was investigated. DNA with thiophosphate groups (DNA[P=S]) obtained by such a way was studied in reactions of hydrolysis and pyrophosphorolysis catalyzed by DNA polymerase I KF. It is shown that the rate of DNA elongation is decreased both on the step of incorporation of dNMP alpha S residues and on the step of incorporation of the next dNMP residue. The rate of pyrophosphorolysis of 3'-terminal dNMP alpha S was demonstrated to be one order of magnitude less in comparison with the corresponding reaction with the natural dNMP residue. Contrary, the rate of 3'----5'-exonuclease hydrolysis of both DNA[P=S] and DNA of the same structure revealed no distinguishable differences.
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PMID:[DNA polymerase I, Klenow fragment of Escherichia coli: hydrolysis and pyrophosphorolysis of DNA containing phosphothioate groups]. 267 72

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.
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PMID:In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I. 270 42

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.
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PMID:Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase. 282 55

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.
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PMID:Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I. 283 46

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.
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PMID:[Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver]. 284 24

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5


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