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)

DNA primases encoded by the conjugative plasmids ColIb-P9 (IncI1), RP4, and R751 (IncP), and the protein of the Escherichia coli satellite phage P4 alpha were shown to contain a common amino acid sequence motif -E-G-Y-A-T-A-. The P4 alpha gene product, required for initiation of phage DNA replication, exhibits primase activity on single-stranded circular DNA templates. This priming activity resembles the enzymatic activity of DNA primases encoded by conjugative plasmids in terms of template utilization and the ability to synthesize primers that can be elongated by DNA polymerase III holoenzyme. The -E-G-Y-A-T-A- motif is part of an extended sequence region most conserved within the primase domains of the four enzymes. Single amino acid substitutions generated in the -E-G-Y-A-T-A- motif of the RP4 TraC2 and the P4 alpha protein affect priming activity, supporting the hypothesis that the conserved sequence motif is part of the active center for primase function. A mutation that eliminates priming activity causes P4 phage to grow poorly and to depend upon the host dnaG primase. Computer analysis identified two additional sequence motifs within the amino acid sequence of the P4 alpha protein: a potential zinc-finger motif and a "type A" nucleotide binding site, both strikingly similar to sequence motifs described in various DNA primases and helicases.
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PMID:A common sequence motif, -E-G-Y-A-T-A-, identified within the primase domains of plasmid-encoded I- and P-type DNA primases and the alpha protein of the Escherichia coli satellite phage P4. 161 4

X-ray studies of the proofreading 3',5'-exonuclease site of the large (Klenow) fragment of DNA polymerase I have detected a binuclear metal complex consisting of a pentacoordinate metal (site A) which shares a ligand, Asp-355, with an octahedral metal (site B) [Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., & Steitz, T. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8924-8928; Beese, L. S., & Steitz, T. A. (1991) EMBO J. 10, 25-33]. Kinetic studies of the activation of the 3',5'-exonuclease reaction by Co2+, Mn2+, or Mg2+, at low concentrations of DNA, reveal sigmoidal activation curves for the three metal ions with Hill coefficients of 2.3-2.4 and K0.5 values of 16.6 microM, 4.2 microM, and 343 microM, respectively. The binding of Co2+ to the enzyme results in the appearance of an intense visible absorption spectrum of the metal ion with maxima at 633, 570, and 524 nm and extinction coefficients of 190, 194, and 150 M-1 cm-1, respectively, suggesting the formation of a pentacoordinate Co2+ complex. Optical titration with Co2+ yields a sigmoidal titration curve which is best fit by assuming the cooperative binding of three Co2+ ions with a K0.5 of 39.9 microM, comparable to the value of 16.6 microM obtained kinetically. Displacement of Co2+ by 1 equiv of Zn2+, which binds tightly to the A site of the 3',5'-exonuclease, shifts the optical spectrum to 524 nm and lowers the extinction coefficient to 30 -1 cm-1, indicative of octahedral coordination.2+ the formation of the binuclear complex.
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PMID:Role of divalent cations in the 3',5'-exonuclease reaction of DNA polymerase I. 165 60

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
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PMID:Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth. 169 88

The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural-functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied.
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PMID:Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 170 12

6-Nitroso-1,2-benzopyrone, an oxidation product of 6-amino-1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn(2+)-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template, consisting of a 17-mer DNA primer annealed to M13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I. Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.
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PMID:Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function. 191 72

The molecular mechanism of the effects of zinc ions against herpes simplex virus (HSV) infection was investigated. Zinc sulphate (100 microM) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1% of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mM-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein-dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.
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PMID:The mechanism of the antiherpetic activity of zinc sulphate. 217 90

A photoactive nucleotide analogue of dUTP, 5-azido-2'-deoxyuridine 5'-triphosphate (5-N3dUTP), was synthesized from dUMP in five steps. The key reaction in the synthesis of 5-N3dUTP is the nitration of dUMP in 98% yield in 5 min at 25 degrees C using an excess of nitrosonium tetrafluoroborate in anhydrous dimethylformamide. Reduction of the resulting 5-nitro compound with zinc and 20 mM HCl gave 5-aminodeoxyuridine monophosphate (5-NH2dUMP). Diazotization of 5-NH2dUMP with HNO2 followed by the addition of NaN3 to the acidic diazonium salt solution gave a photoactive nucleotide derivative in 80-90% yield. The monophosphate product was identified as 5-N3dUMP by proton NMR, UV, IR, and chromatographic analysis as well as by the mode of synthesis and its photosensitivity. After formation of 5-N3dUTP through a chemical coupling of pyrophosphate to 5-N3dUMP, the triphosphate form of the nucleotide was found to support DNA synthesis by Escherichia coli DNA polymerase I at a rate indistinguishable from that supported by dTTP. When UMP was used as the starting compound, 5-N3UTP was formed in an analogous fashion with similar yields and produced a photoactive nucleotide which is a substrate for E. coli RNA polymerase. To prepare [gamma-32P]-5-N3dUTP for use as an active-site-directed photoaffinity labeling reagent, a simple method of preparing gamma-32P-labeled pyrimidine nucleotides was developed. [gamma-32P]-5-N3dUTP is an effective photoaffinity labeling reagent for DNA polymerase I and was found to bind to the active site with a 2-fold higher affinity than dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. 354 18

Bis(diphenylphosphine)ethane (DPPE) and its bis[chlorogold(I)] [DPPE(Au2Cl2)], and bis[trichlorogold(III)] [DPPE(Au2Cl6)], complexes have in vivo antitumor activity. To determine if interaction with metals in situ can play a role in the antitumor activity of DPPE, we have studied the effects of DPPE, DPPE(Au2Cl2), DPPE(Au2Cl6) and mixtures of DPPE with metal salts on in vitro and in vivo biological systems. The in vitro cytotoxic potencies of the two DPPE-gold complexes were approximately 10-fold greater than that of DPPE. In addition, the cytotoxic potency of DPPE was increased when incubated with cells in the presence of Au(III) and Cu(II) salts, whereas Mg(II), Zn(II), Mn(II), Fe(II), Co(II), and Cd(II) had no effect. The effects of DPPE, DPPE(Au2Cl2) and mixtures of DPPE and metal salts on the activity of a model enzyme system, DNA polymerase alpha were measured. While DPPE did not inhibit the activity of DNA polymerase alpha, the DPPE(Au2Cl2) complex and mixtures of DPPE and Cu(II) salts inhibited the activity of the enzyme. Consistent with the effects observed in vitro, coadministration of Cu(II) or Au(III) increased the in vivo potency of DPPE in mice bearing i.p. P388 leukemia. Fifteen other DPPE analogues were evaluated for in vivo antitumor activity and for the effect of Cu(II) on their in vitro cytotoxic potency; there was a relationship between the ability of Cu(II) to potentiate the cytotoxic activities of DPPE analogues and their having in vivo antitumor activity.
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PMID:Modulation of the antitumor and biochemical properties of bis(diphenylphosphine)ethane with metals. 375 63

The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.
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PMID:Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. 388 92

Evidence is presented that DNA polymerase of avian myeloblastosis virus has an obligatory zinc requirement for activity. Previous studies indicate that the purified polymerase contains zinc in a stoichiometry of about 1 g-atom/mole. We now find that the enzyme-bound zinc is exchangeable with radioactive (65)Zn; after isoelectric focusing, the radioactive (65)Zn is coincident with polymerase activity. Dialysis of the (65)Zn-labeled polymerase against the chelator, 1,10-phenanthroline, results in a progressive loss of radioactive (65)Zn and polymerase activity. Thereupon, incubation of the inactivated enzyme with Zn(2+) fully restores activity. Thus, the DNA polymerase present in an oncogenic RNA virus, like animal DNA polymerases, can be rigorously classified as a zinc metalloenzyme. DNA polymerase of avian myeloblastosis virus is inactivated by 1,10-phenanthroline at a much faster rate than the bacterial and animal DNA polymerases that have been tested. It may, therefore, be possible to inactivate selectively DNA polymerases from animal tumor viruses by brief exposure to appropriate metal chelators.
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PMID:Reverse transcriptase: correlation of zinc content with activity. 414 May 13


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