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)

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.
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PMID:Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps. 240 6

A modified assay of nick-translation of nuclei has been developed to study the chromatin structure of human beta-like globin genes in nuclei of K 562 cell line. Nuclei were gently digested with DNase I and nick-translated with E. coli DNA polymerase I in the presence of 32P-triphosphate nucleotides. The total DNA from the labelled nuclei was used as probes to hybridize restricted fragments of beta-like globin genes which have been immobilized on Diazobenzyloxymethyl (DBM) paper. Using this approach we have observed that in K 562 nuclei all beta-like globin genes, including epsilon, gamma, delta, and beta-globin genes and human 18 S ribosomal genes are preferentially labelled in comparison to alpha-lactalbumin and c-sis genes which do not express in K 562 cells, but the total DNA from nick-translated nuclei of a nonerythroid cell line hybridized none of those genes except for 18 S ribosomal gene. This assay is a simple and fast method for surveying chromatin structure of any individual DNA sequence in nuclei once the corresponding clone is available.
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PMID:A study of the chromatin structure of human beta-like hemoglobin genes in K562 cell line with a modified assay of nick-translation of nuclei. 262 96

The 10 distinctive polypeptides of DNA polymerase III holoenzyme, purified as individual subunits or complexes, could be reconstituted to generate a polymerase with the high catalytic rate of the isolated intact holoenzyme. Functions and interactions of the subunits can be inferred from partial assemblies of the pol III core (alpha, epsilon, and theta subunits) with auxiliary subunits. The core possesses the polymerase and proofreading activities; the auxiliary subunits provide the core with processivity, the capacity to replicate long stretches of DNA without dissociating from the template. In a sequence of reconstruction steps, the beta subunit binds the primed template in an ATP-dependent manner through the catalytic action of a complex made up of the gamma, delta, delta', chi, and psi polypeptides. With the beta subunit in place, a processive polymerase is produced upon addition of the core. When the tau subunit is lacking, binding of polymerase to the primed template is less efficient and stable. The tau-less reconstituted polymerase is more prone to dissociation upon encountering secondary structures in the template in its path, such as a hairpin region in the single strand or a duplex region formed by a strand annealed to the template. With the tau subunit present, the interaction of the core.beta complex (the basic unit of a processive polymerase) with the primed template is strengthened. The tau-containing reconstituted polymerase can replicate DNA continuously through secondary structures in the template. The two distinctive kinds of processivity demonstrated by the tau-less and tau-containing reconstituted polymerases fit nicely into a scheme in which, organized as an asymmetric dimeric holoenzyme, the tau half is responsible for continuous synthesis of one strand, and the less stable half for discontinuous synthesis of the other.
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PMID:DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits. 328 27

We have previously shown the presence of sphingomyelin and sphingomyelinase in cell nuclei, suggesting that they may play a role in the intranuclear production of sphingosine, a potent bioactive molecule modulating diverse cellular functions. In the present study, the direct effects of sphingosine (C18:1) on the activity of DNA replication/repair polymerases were studied in vitro. Sphingosine had no effect on DNA polymerases alpha and beta and slightly inhibited DNA polymerases gamma, delta, and epsilon. In contrast, sphingosine strongly inhibited the activity of primase in a dose-dependent manner. On the other hand, dihydrosphingosine (C18:0), glycolipids, sphingomyelin, and ceramide had no effect on primase activity. Sphingosine equally inhibited the activity of primase complexed with DNA polymerase alpha, as well as its free form, with a Ki value of 4 microM. A gel-retardation analysis showed that the binding of primase with 32P-labeled template DNA was suppressed by sphingosine. Inhibition by sphingosine was competitive with the DNA template, but not with the substrate NTPs. After product analysis, a dose-dependent decrease in the amount of RNA primer products, consisting mainly of 10- and 11-mers, was observed in the presence of sphingosine, indicating that it inhibits the synthesis of RNA primers by primase. Sphingosine, however, had no effect on T7 RNA polymerase.
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PMID:Sphingosine inhibits the synthesis of RNA primers by primase in vitro. 751 54

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

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

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

A DNA polymerase with a 3'-to 5'-exonuclease that copurified with polymerase-primase from calf thymus was purified and extensively characterized. Its exonuclease degraded single-stranded DNA from 3' to 5' in a strictly distributive manner. On synthetic template-primer junctions, 3'-terminal mispairs were excised with a 10- to 20-fold preference over correctly paired nucleotides. In comparison to the 3'- to 5'-exonuclease the DNA polymerase activity was rather low. The ratio of nucleotides incorporated to nucleotides excised was in the order of 1 to 3 nucleotide insertions per excision, suggesting that net forward DNA synthesis is not the primary role of this DNA polymerase. DNA synthesis was performed with a low processivity in the presence and absence of PCNA. Both the polymerase and exonuclease activities were inhibited to a comparable extent by AMP. Thus, the exonuclease-polymerase might represent a novel DNA polymerase that we tentatively designate as DNA polymerase zeta. Possible benefits of DNA polymerase zeta in the process of error correction and the apparent dichotomy of an built-in proofreading activity for the processive DNA polymerases gamma, delta, and epsilon and an obviously external proofreading function for the less processive animal cell DNA polymerases alpha and beta are discussed.
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PMID:An error-correcting proofreading exonuclease-polymerase that copurifies with DNA-polymerase-alpha-primase. 838 85

The gamma complex subassembly (gamma delta delta' chi psi) of DNA polymerase III holoenzyme couples ATP to assemble the ring-shaped beta subunit around DNA forming a DNA sliding clamp. This beta clamp is needed for highly processive synthesis by the holoenzyme. Here, the delta and delta' subunits of the gamma complex are studied for their structural and functional interaction with each other and with the gamma subunit. Both delta and delta are monomeric in their native state, and they bind each other tightly to form a 1:1 complex. Neither delta nor delta' alone binds tightly to the gamma subunit. However, as a complex, delta delta' binds gamma tightly to form a gamma delta delta' complex. The fact that all three subunits, gamma, delta, and delta', are needed to form a tight complex correlates well with activity assays which show that gamma and delta are capable but inefficient in assembly of the beta ring onto DNA and delta' is needed for an efficient reaction.
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PMID:DNA polymerase III accessory proteins. II. Characterization of delta and delta'. 850 4

DNA polymerase activity was detected and characterized in nuclear extracts from trophozoites of Entamoeba histolytica. The activity was high at pH 2 to pH 6, but at pH 8 and 10 the activity was very low. The presence of K+ was inhibitory for the activity and a higher concentration of K+ markedly inhibited the activity. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 to 8 mM. The activity was markedly inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases alpha, delta, and epsilon and also by N-ethylmaleimide which is an inhibitor of DNA polymerases, alpha, gamma, delta and epsilon. However, inhibition of the activity by 2', 3'-dideoxythymidine-5'-triphosphate which is an inhibitor of DNA polymerases beta and gamma was relatively weak. Thus sensitivity of the E. histolytica enzyme to these inhibitors was similar to that of mammalian DNA polymerases (alpha, delta and epsilon) of the alpha family. Monoclonal antibodies against human DNA polymerase alpha did not bind to DNA polymerase of E. histolytica.
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PMID:Detection and characterization of DNA polymerase activity in Entamoeba histolytica. 882 52


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