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 avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.
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PMID:Avian retrovirus pp32 DNA-binding protein. I. Recognition of specific sequences on retrovirus DNA terminal repeats. 629 95

Relative to nonreplicating DNA in mature simian virus 40 (SV40) chromosomes, newly synthesized DNA in replicating SV40 chromosomes was found to be hypersensitive to the nonspecific endonucleases, micrococcal nuclease (MNase), DNase I, and DNase II. Nascent DNA, pulse labeled in either intact cells or nuclear extracts supplemented with cytosol, was digested about 5-fold faster and about 25% more extensively than uniformly labeled DNA in mature viral chromosomes. Pulse-chase experiments in vitro revealed a time-dependent chromatin maturation process that involved two distinct steps: (i) conversion of prenucleosomal DNA (PN-DNA) into immature nucleosomal oligomers and (ii) maturation of newly assembled chromatin into a structure with increased nuclease resistance. PN-DNA was hypersensitive to MNase, releasing short DNA fragments which were subsequently solubilized by the nuclease. However, when the nascent PN-DNA was specifically removed by digestion of replicating viral chromosomes with Escherichia coli exonuclease III (3'-5') and phage T7 exonuclease (5'-3'), subsequent digestion of the remaining chromatin with MNase revealed the same degree of hypersensitivity observed prior to exonuclease treatment. Furthermore, newly assembled nucleosomal oligomers, isolated after a brief MNase digestion of replicating viral chromosomes, were also hypersensitive to MNase relative to oligomers isolated from mature chromosomes. Hybridization analysis of the DNA in these immature oligomers revealed that it originated from both sides of replication forks. Inhibition of DNA polymerase alpha by aphidicolin inhibited conversion of PN-DNA into nucleosomes but did not inhibit loss of nucleosomal hypersensitivity to MNase. In contrast, components in the soluble fraction of the subcellular system ("cytosol") were required for both DNA replication and chromatin maturation. Analysis of the nucleoprotein products from a MNase digestion of replicating and mature SV40 chromosomes failed to detect a change in nucleosome structure that corresponded to the loss of nuclease hypersensitivity. However, the results presented demonstrate that both PN-DNA and newly assembled immature chromatin, present on both arms of SV40 replication forks, contribute to the commonly observed hypersensitivity of newly replicated chromatin to endonucleases.
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PMID:Structure of chromatin at deoxyribonucleic acid replication forks: nuclease hypersensitivity results from both prenucleosomal deoxyribonucleic acid and an immature chromatin structure. 631 Dec 55

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.
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PMID:Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks. 636 Oct 28

Essentially all of the DNA polymerase alpha activity in CV-1 monkey cells could be extracted as an enzyme complex that used DNA substrates with a low primer:template ratio, such as denatured DNA, at least 25 times more efficiently than did purified alpha polymerase. This form of the enzyme was rapidly dissociated either by the nonionic detergent Triton X-100 or by chromatography on phosphocellulose to generate alpha polymerase and its protein cofactor complex, C1C2. Both alpha polymerase and C1C2 were then independently purified free of deoxyribonuclease, RNA polymerase, DNA ligase, and ATPase activities, and the C1C2 complex was shown to consist of at least two proteins. Purified C1C2, which exhibited no DNA polymerase activity, completely restored the ability of alpha polymerase to use denatured DNA. Although high concentrations of denatured DNA inhibited the activity of C1C2, which binds tightly to single-stranded but not double-stranded DNA, low concentrations catalyzed reconstitution of alpha polymerase with C1C2. The resulting enzyme complex was chromatographically distinct from alpha polymerase on DEAE-Bio-Gel, was no longer dependent upon addition of C1C2 in order to utilize denatured DNA as effectively as DNase I-activated DNA, and was not inhibited by high concentrations of denatured DNA. These properties of the purified reconstituted enzyme were indistinguishable from those native alpha X C1C2-polymerase.
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PMID:Preparation of DNA polymerase alpha X C1C2 by reconstituting DNA polymerase alpha with its specific stimulatory cofactors, C1C2. 688 71

Chicken erythrocyte nuclei have been labeled in the active regions of the chromosome by using the nick translation reaction. In this procedure, accessible areas of the genome are preferentially nicked by the action of pancreatic DNase I and subsequently labeled by using DNA polymerase I from Escherichia coli. These nuclei were employed as a substrate for studying the factors responsible for maintaining the special chromatin conformation of the overall population of active genes. Treatment of nuclei with 0.35 M NaCl resulted in the loss of DNase I sensitivity in the active genes, but this sensitivity could be restored when nuclei were reconstituted with the NaCl eluate. Further purification of the released factors revealed that the HMG (high-mobility group) proteins HMG-14 and HMG-17 are involved in maintaining the conformation of the active regions. These factors are not tissue specific and seem to be involved in the chromosomal structure of most of the active genes.
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PMID:Reconstitution of a deoxyribonuclease I-sensitive structure on active genes. 692 20

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.
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PMID:Interactions of calf thymus DNA polymerase alpha with primer/templates. 747 73

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.
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PMID:Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization. 752 8

Replication complexes containing wild-type and RNase H-deficient p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) were analyzed by DNase I and S1 footprinting. While crystallography and chemical footprinting data demonstrate that 15-18 bases of primer and template occupy the DNA polymerase and RNase H active centers, enzymatic footprinting suggests that a larger portion of substrate is encompassed by the replicating enzyme. Independent of the position of DNA synthesis arrest, template nucleotides +7 to -23 and primer nucleotides -1 to -25 are nuclease resistant. On both DNA strands, position -20 remains accessible to DNase I cleavage, suggestive of an alteration in nucleic acid structure between exiting the RNase H catalytic center and leaving the C-terminal p66 domain. A model of HIV-1 RT containing an extended single-stranded template and duplex region was constructed on the basis of the structure of an RT/DNA complex. Mapping of footprint data onto this model shows consistency between biochemical and structural data, implicating a contribution from domains proximal to the catalytic centers.
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PMID:An expanded model of replicating human immunodeficiency virus reverse transcriptase. 753 89

Escherichia coli DNA polymerase III holoenzyme in the presence of ATP and E. coli single-stranded DNA-binding protein forms an initiation complex on a primed template capable of rapid and highly processive DNA replication. DNase I digestion of initiation complexes demonstrated that holoenzyme protected 27-30 nucleotides of primer. Like the formation of initiation complexes, this protection required both ATP and E. coli single-stranded DNA-binding protein. Initiation complexes assembled with core DNA polymerase III (alpha, epsilon, and theta subunits), gamma-complex (gamma, delta, delta', chi, and omega) and the beta subunit produced a footprint identical to that formed with intact holoenzyme, indicating that initiation complexes formed with reconstituted enzyme and those formed with holoenzyme were equivalent. The presence of the tau subunit in reconstituted initiation complexes did not alter the DNase I footprint. Preinitiation complexes (gamma-complex plus beta subunit) assembled onto primer-template in an ATP-dependent reaction protected a larger region of the primer than did holoenzyme. The addition of core DNA polymerase III to preintiation complexes restored the 30-nucleotide footprint observed with intact holoenzyme. These results suggest that holoenzyme subunits rearrange during initiation complex formation.
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PMID:Escherichia coli DNA polymerase III holoenzyme footprints three helical turns of its primer. 780 36

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.
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PMID:Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template. 803 92


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