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)

Carbocylic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which may be useful in the treatment of AIDS. We have synthesized the 5'-triphosphate of Carbovir and examined its ability to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49) and other retroviral reverse transcriptases, as well as human DNA polymerases alpha, beta, gamma (EC 2.7.7.7) and DNA primase (EC 2.7.7.6). Carbovir triphosphate emerges as a highly selective inhibitor of reverse transcriptases with little, if any, effect on the cellular enzymes. 3'-Azido-2',3'-dideoxythymidine (AZT) triphosphate and the two dideoxynucleoside triphosphates, ddTTP and ddGTP, inhibited HIV-1 reverse transcriptase to the same degree as Carbovir triphosphate, but were less selective in that they also inhibited DNA polymerases beta and gamma. We conclude that Carbovir is a highly selective antiretroviral agent.
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PMID:Comparison of the effect of Carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human polymerases. 247 36

An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n. Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end. The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end. With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template. A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA. The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin. Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template. This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA. The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E. coli DNA polymerase I Klenow fragment. Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).
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PMID:DNA primase and the replication of the telomeres in Oxytricha nova. 247 56

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.
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PMID:A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae. 252 82

Polyomavirus DNA replication is normally restricted to rodent cells, and simian virus 40 (SV40) DNA replication is restricted to primate cells. We demonstrate that DNAs containing the polyomavirus origin can be replicated in monkey cells which constitutively express SV40 large T antigen. Permissivity is most likely caused by SV40 T antigen modification of cellular protein(s) required to replicate the polyomavirus origin. A possible target for the T-antigen-induced modification is DNA polymerase alpha-DNA primase.
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PMID:Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication. 255 68

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.
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PMID:Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae. 255 94

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.
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PMID:Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification of the associated kinase. 256 5

DNA primase-DNA polymerase alpha, purified 53,000-fold from CV-1 cells, synthesized predominantly (p)ppA(pA)6-primed DNA on a poly(dT) template. About 80% of the RNA primers synthesized on an M13 DNA template were (p)ppA/G(pN)5-7, and 20% were (p)ppA/G(pN)0-4. RNA primer size was determined by gel electrophoresis after removing nascent DNA with phage T4 DNA polymerase 3'-5' exonuclease, leaving a single dNMP at the 3'-end of the RNA primer, and the terminal 5'-(p)ppN residue was determined by "capping" with [alpha-32P]GTP using vaccinia guanylyl-transferase. The processivity of DNA synthesis initiated by de novo synthesis of RNA primers was the same as that initiated on pre-existing RNA primers (10-15 dNMPs), although initiation on pre-existing primers was strongly preferred. Primers always began with A or G, even at high levels of CTP or UTP, although the ratio of A to G varied from 4:1 to 1:1 depending on the relative concentrations of ATP and GTP in the assay. ATP and GTP had no effect on primer length, but the fraction of shorter RNA primers increased 2-fold with higher concentrations of CTP or UTP. Nearest-neighbor analysis revealed a preference for purine ribonucleotides at RNA covalently linked to the 5'-end of DNA (RNA-p-DNA) junctions, and increasing the concentration of a single rNTP increased slightly its presence at RNA-p-DNA junctions. Thus, the base composition and size of RNA primers synthesized by DNA primase-DNA polymerase alpha is modulated by the relative concentrations of ribonucleoside triphosphates.
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PMID:DNA primase-DNA polymerase alpha from simian cells. Modulation of RNA primer synthesis by ribonucleoside triphosphates. 258 49

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.
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PMID:DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA. 258 40

Nucleoids, prepared by salt extraction of non-DNase-digested nuclei, have properties similar, but not identical, to those of nuclear matrices which are prepared by salt extraction of DNase-digested nuclei. Nuclear matrices retained less pulse-labelled DNA, slightly less bound DNA polymerase alpha and DNA primase, but had greater in vitro DNA synthesis and in vitro priming. Nucleoids contained larger (110 S) DNA chains than nuclear matrices (30 S). Each type of residual nuclear structure could synthesize 4.5 S Okazaki fragments. When extracted with increasing concentrations of salt, DNase-digested nucleo lost the ability for further elongation of the 4.5 S DNA intermediate after 0.1-0.2 M NaCl, whereas undigested nuclei retained this ability up to 0.9 M NaCl. Chain elongation to 28 S DNA chains could be restored to nucleoids, but not to nuclear matrices, by the addition of nuclear extracts.
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PMID:Nucleoids, a subnuclear system capable of chain elongation. 259 77

The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.
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PMID:Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. 264 67


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