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 phage P22 gene 12 protein was found to be like the Escherichia coli dnaB protein in that it stimulated phiX174 DNA synthesis in heat-inactivated extracts of dnaB temperature-sensitive cells (see preceding paper, Wickner, S. (1984) J. Biol. Chem. 259, 14038-14043). phiX174 replication catalyzed by the purified P22 12 protein also by-passed the normal requirement for dnaC protein. However, synthesis still required dnaG primase and the DNA polymerase III holoenzyme components. This DNA synthesis reaction has been reconstituted with purified proteins and found to require P22 12 protein, dnaG protein, DNA polymerase III holoenzyme components, 4 dNTPs, Mg2+, any one of ATP, GTP, UTP, or CTP and single-stranded DNA. The reaction has been dissected into partial reactions: (a) in a prepriming reaction, P22 12 protein binds to single-stranded DNA in an ATP-dependent reaction (Wickner, S. (1984) J. Biol. Chem. 259, 14038-14043); (b) in a priming reaction requiring at least one rNTP and the other dNTPs or rNTPs, dnaG primase catalyzes oligonucleotide synthesis dependent on the P22 12 protein-DNA complex; (c) finally, DNA polymerase III holoenzyme components catalyze DNA elongation of the primer.
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PMID:Oligonucleotide synthesis by Escherichia coli dnaG primase in conjunction with phage P22 gene 12 protein. 623 58

The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.
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PMID:Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study. 637 96

The triphosphates of acyclovir (ACV), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and E-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) have been examined for their inhibitory effects on the endogenous DNA polymerase reactions of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV). All three triphosphates (ACVTP, FIACTP and BVdUTP) inhibited the HBV and WHV DNA polymerases by competing with the corresponding natural substrates. FIACTP was the most potent inhibitor of HBV and WHV DNA polymerase while ACVTP was the least effective inhibitor. The inhibitory properties of these compounds were compared with those of the 5'-triphosphates of 1-beta-arabinofuranosyl-cytosine (ara-CTP) and 1-beta-arabinofuranosylthymine (ara-TTP). The 50% inhibitory doses for HBV and WHV DNA polymerases were in the following order: FIACTP less than BVdUTP less than ara-TTP less than ACVTP less than ara-CTP. BVdUTP appeared to be an efficient alternate substrate to dTTP for HBV DNA polymerase while FIACTP was much less efficient when substituted for dCTP. ACVTP did not act as an alternate substrate to dGTP and appeared to prevent DNA chain elongation.
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PMID:Inhibition of human and woodchuck hepatitis virus DNA polymerase by the triphosphates of acyclovir, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine and E-5-(2-bromovinyl)-2'-deoxyuridine. 654 55

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.
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PMID:Synergistic inhibition of human leukemia cell growth by deoxyguanosine and 1-beta-D-arabinofuranosylcytosine. 671 15

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.
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PMID:Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells. 675 37

Preparations of DNA polymerase alpha from early embryos of Drosophila melanogaster catalyze the ATP-dependent synthesis of DNA with single-stranded M13 DNA or poly(dT) templates. In the case of M13 DNA, GTP, but not UTP or CTP, can replace ATP. The reaction is completely dependent on added template and is not inhibited by alpha-amanitin. Alkaline hydrolysis of the product synthesized in the presence of [alpha-32P]dATP and poly(dT) generates 32P-labeled 3'(2') adenylate, showing that a covalent ribo-deoxynucleotide linkage is formed. Furthermore, incorporation of ribonucleotides occurs at the 5' end of the newly synthesized polynucleotide chain. These findings are consistent with the hypothesis that a ribo-oligonucleotide primer is synthesized by primase action and subsequently elongated by DNA polymerase. Under the appropriate conditions, DNA polymerase I from Escherichia coli can elongate primers formed by primase in the presence of ATP and poly(dT). Primase activity copurifies with DNA polymerase alpha and may be part of the multisubunit polymerase molecule.
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PMID:A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. 680 12

An enzymatic activity that synthesizes oligoribonucleotides in lengths of 9-10 nucleotides and multiples thereof has been purified over 10,000-fold from mouse hybridoma cells. The oligoribonucleotides serve as primers to initiate DNA synthesis, and the activity has properties expected of mammalian DNA primase. The most highly purified fraction has two major protein components, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, of 56,000 and 46,000 Da. These proteins co-purify in a 1:1 stoichiometry along with oligoribonucleotide synthesis activity and with an activity that initiates the synthesis of DNA by DNA polymerase alpha. The sedimentation coefficient on glycerol gradients is 5.5 S, and this is consistent with one 56,000- and one 46,000-Da subunit/native enzyme. No DNA polymerase activity was detected in the most highly purified fraction. Poly(dIT) is the most active template, whereas a variety of single-stranded DNA templates are 10-15% as active and double-stranded DNA templates are 10-15% as active and double-stranded DNA is less than 1%. rATP is an absolute requirement as is Mg2+. No ATPase activity was detected with or without addition of DNA, single- or double-stranded. (NH4)2SO4 and NaPO4 buffer, pH 7.6, are inhibitory above 20 mM, whereas KCl is inhibitory above 80 mM. beta-D-arabinose-CTP is a strong inhibitor of primase; approximately 50% inhibition is observed when present at one-fifth the concentration of rCTP.
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PMID:A DNA primase from mouse cells. Purification and partial characterization. 688 74

One of the two forms of DNA polymerase alpha from ovaries of the frog Xenopus laevis catalyzed ribonucleoside triphosphate-dependent DNA synthesis on single-stranded circular fd phage DNA templates. DNA synthesis was dependent on ATP and added template. CTP, GTP, and UTP stimulated DNA synthesis but were not required and could not substitute for ATP. DNA synthesis was not inhibited by alpha-amanitin. Neither poly(dT) nor double-stranded DNA served as template. Analysis of [32P]-dTMP-labeled product by neutral and alkaline agarose gel electrophoresis showed that 0.1- to 1-kilobase DNA fragments (average size of approximately equal to 0.25 kilobase) were synthesized. The fragments were not covalently linked to the template. Either [alpha-32P]NMP, [gamma-32P]ATP, or [gamma-32P]GTP were incorporated also into the product. Analysis of the product after hydrolysis by KOH, alkaline phosphatase, or bacteriophage T4 3' leads to 5' exonuclease showed the presence of a small oligoribonucleotide primer at the 5' end of the newly synthesized DNA. NTP-dependent DNA-synthesizing activity copurified on six columns and cosedimented during glycerol gradient centrifugation with one form of DNA polymerase alpha activity but not with the other form. These results suggest that DNA primase activity is associated with one of the two forms of X. laevis DNA polymerase alpha.
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PMID:DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries. 696 3

Cytosine arabinoside (Ara-C) is a very potent inhibitor of DNA synthesis in mammalian cells. This inhibition does not appear to be due to the direct inhibition of DNA polymerase by Ara-CTP (triphosphate of Ara-C) because it is a very weak competitive inhibitor of this enzyme. The inhibition of DNA synthesis appears to be due to the incorporation of Ara-C into DNA producing chain termination of polydeoxynucleotide elongation.
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PMID:Biochemical pharmacology of cytosine arabinoside. 716 66

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia. 747 75


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