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 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.
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PMID:Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form. 694 51

Using mammalian DNA polymerases alpha, beta and terminal deoxynucleotidyl transferase, we have examined the inhibitory action of X-irradiated DNA on in vitro DNA synthetic activities of these enzymes. It was found that DNA polymerase beta was highly sensitive inhibition by the irradiated DNA as well as DNA polymerases I of E. coli, while DNA polymerase alpha was at least two hundred times more resistant to inhibition than DNA ploymerase beta. Terminal deoxynucleotidyl transferase was inhibited moderately by the single-stranded form of the irradiated DNA. Since the inhibition was competitive with respect to a template-initiator for all DNA polymerases or an initiator for terminal deoxynucleotidyl transferase, the differences in sensitivities to the inhibition may be due to the different affinities of the enzymes to the X-ray-induced inhibitory sites on the DNA strand.
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PMID:Differential inhibition of mammalian DNA polymerases by X-irradiated DNA. 700 27

Three DNA polymerases were partially purified from the embryos, liver and mitochondria of the loach Misgurnus fossilis by DEAE- and phosphocellulose chromatography and were identified as DNA polymerases alpha, beta and gamma. DNA polymerase alpha prefers the activated DNA as a template-primer and has a sedimentation value of 6.8S. The enzyme is stimulated by 50 mM potassium phosphate, KCl and, to a lesser extent, NaCl; has a pH optimum of 7.5 and is sensitive to N-ethylmaleimide. DNA polymerase beta also prefers the activated DNA as a template-primer but shows a sedimentation coefficient of 3.0 S. The enzyme is inhibited by salts and has a pH optimum of 8-9; its activity is rather resistant to N-ethylmaleimide. DNA polymerase gamma has a sedimentation value of 6.3S, shows a high activity on poly(A) . oligo(dT) in the presence of 100 mM potassium phosphate and a lesser activity in the presence of 150 mM KCl and NaCl. The enzyme has a pH optimum of 7.0.
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PMID:[Isolation and characterization of DNA polymerase alpha, beta and gamma from the cells of the loach (Misgurnus fossilis)]. 729 16

Inhibitory effects of 2'-deoxy-L-thymidine 5'-triphosphate (L-dTTP), the enantiomer of the natural substrate D-dTTP, on the activity of mammalian DNA polymerases alpha, beta and gamma, Escherichia coli DNA polymerase I and human immunodeficiency virus 1 (HIV-1) reverse transcriptase were examined. When poly(rA)n-oligo(dT)12-18 was used as the template-primer, L-dTTP showed remarkable inhibitory effect on HIV-1 reverse transcriptase in competitive fashion with respect to the substrate dTTP. In contrast, L-dTTP did not inhibit DNA polymerases alpha and was slightly inhibitory to DNA polymerase beta. These results suggest that the nuclear DNA polymerases alpha and beta showed high specificity for the substrate with the natural configuration of the sugar moiety, D-dTTP, exhibiting little or no ability to recognize L-dTTP, whereas HIV-1 reverse transcriptase essentially lacked the ability to differentiate the D- and L-sugar moieties.
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PMID:Chiral discrimination of enantiomeric 2'-deoxythymidine 5'-triphosphate by HIV-1 reverse transcriptase and eukaryotic DNA polymerases. 751 92

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.
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PMID:Further characterization of HeLa DNA polymerase epsilon. 753 91

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.
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PMID:Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns. 754 38

5'-Triphosphates of beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-5-fluorocytidine (FddC), 1,3-dioxolane-cytidine (OddC), and 1,3-dioxolane-5-fluorocytidine (FOddC) were evaluated as inhibitors and substrates for human DNA polymerases alpha, beta, gamma, delta, and epsilon. L-ddCTP was not a substrate or inhibitor for any DNA polymerase studied; L-FddCTP was not an inhibitor or substrate for replicative DNA polymerases and was a less potent inhibitor of DNA polymerases gamma and beta than its D-enantiomer by 2 orders of magnitude. In contrast, all L-dioxolane analogs were potent inhibitors and chain terminators for all cellular DNA polymerases studied. The Ki values of their 5'-triphosphates for DNA polymerase gamma were found to be in the following order: D-ddC < D-FddC L-OddC D-FOddC < L-FOddC << L-FddC. The Ki values of L-OddCTP for the reactions catalyzed by DNA polymerases alpha, delta, epsilon, beta, and gamma were 6.0, 1.9, 0.4, 3.0, and 0.014 microM, respectively, and those of L-FOddCTP were 6.5, 1.9, 0.7, 19, and 0.06 microM, respectively. The Km values for incorporation of L-OddCTP into the standing points of primer extension were also evaluated and determined to be 1.3, 3.5, 1.5, 2.8, and 0.7 microM for DNA polymerases alpha, delta, epsilon, beta, and gamma, respectively. The incorporation of dioxolane analogs into DNA by replicative DNA polymerases could explain their potent cellular toxicity.
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PMID:L- and D-enantiomers of 2',3'-dideoxycytidine 5'-triphosphate analogs as substrates for human DNA polymerases. Implications for the mechanism of toxicity. 755 45

We have examined the capacity of calf thymus DNA polymerases alpha, beta, delta, and epsilon to perform in vitro translesion synthesis on a substrate containing a single d(GpG)-cisplatin adduct placed on codon 13 of the human HRAS gene. We found that DNA synthesis catalyzed by DNA polymerases alpha, delta, and epsilon was blocked at the base preceding the lesion. Addition of proliferating cell nuclear antigen to DNA polymerase delta and replication protein A to DNA polymerase alpha did not restore their capacity to elongate past the adduct. On the other hand, DNA polymerase beta efficiently bypassed the cisplatin adduct. Furthermore, we observed that DNA polymerase beta was the only polymerase capable of primer extension of a 3'-OH located opposite the base preceding the lesion. Likewise, DNA polymerase beta was able to elongate the arrested replication products of the other three DNA polymerases, thus showing its capacity to successfully compete with polymerases alpha, delta, and epsilon in the stalled replication complex. Our data suggest (i) a possible mechanism enabling DNA polymerase beta to bypass a d(GpG)-cisplatin adduct in vitro and (ii) a role for this enzyme in processing DNA damage in vivo.
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PMID:DNA polymerase beta bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene. 777 11

Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the alpha, beta, and gamma subunits formed ATP-dependent contacts with the primer-template. The alpha-polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The gamma subunit contacted the primer at positions -13, -18, and -22, with the strongest gamma-primer interactions occurring at position -18. The beta subunit predominated in cross-linking at position -22. Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by gamma at -18 and beta at -22, and the gamma subunit remains a part of the initiation complex, bridging the alpha and beta subunits. Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (gamma-complex (gamma-delta-delta'-chi-psi) and beta subunit) revealed the gamma subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of gamma by alpha at the primer terminus. These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.
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PMID:Escherichia coli DNA polymerase III holoenzyme subunits alpha, beta, and gamma directly contact the primer-template. 789 Jun 80

Halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge, inhibited the activity of eukaryotic DNA polymerases in varying degrees; the Ki values for DNA polymerases, alpha, beta, delta and epsilon were 1.3, 80, 17.5 and 2.0 microM, respectively, whereas it was less effective against E. coli DNA polymerase I. The inhibition occurred competitively with each of dATP and dTTP, but non-competitively with dCTP, dGTP and the template DNA. Thus, halenaquinol sulfate is demonstrated to be a potential inhibitor of DNA polymerases alpha and epsilon, and be a useful tool for analyzing the dNTP binding sites of DNA polymerases.
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PMID:Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge. 807 May 73


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