Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene 4 protein of bacteriophage T7 is both a primase and a helicase. In this paper, we present a detailed description of a third activity, single-stranded DNA-dependent nucleoside 5'-triphosphate hydrolysis, and show that this activity is coupled to the unidirectional translocation of the gene 4 protein on single-stranded DNA (Tabor, S., and Richardson, C.C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 205-209). The competitive inhibitor of NTP hydrolysis, beta, gamma-methylene dTTP, is also a potent inhibitor of gene 4 protein-dependent, RNA-primed DNA synthesis; inhibition is not due to a direct inhibition of T7 DNA polymerase or RNA primer synthesis. We conclude that the energy derived from the hydrolysis of NTPs by the gene 4 protein is required for translocation of the protein to primase recognition sites. Measurement of the rates of hydrolysis of NTPs using a variety of DNAs of known structure and length support the unidirectional translocation of the gene 4 protein on single-stranded DNA. Duplex DNA, RNA, and single-stranded DNA coated with single-stranded DNA-binding protein do not serve as effectors for the nucleoside triphosphatase of the gene 4 protein. Kinetic data suggest that the gene 4 protein does not remain bound to newly synthesized oligoribonucleotide primers but continues to search for other primase recognition sites. Although all the predominant naturally occurring NTPs except rCTP are hydrolyzed by the gene 4 protein, the enzyme shows specificity for dTTP with a Km of 0.4 mM. In the accompanying paper (Matson, S.W., Tabor, S., and Richardson, C.C. (1983) J. Biol. Chem. 258, 14017-14024), we show that the hydrolysis of NTPs is also required for the protein to function as a helicase in duplex regions of DNA.
 ...
PMID:DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7. 613 75

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.
 ...
PMID:DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries. 696 3

Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.
 ...
PMID:Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase. 754 35

3'-Mercapto-2',3'-dideoxy-NTP were synthesized and tested as DNA chain terminating nucleotides. It is shown that the analogues selectively and irreversibly terminate DNA chain elongation by AMV and HIV reverse transcriptases and terminal deoxynucleotidyl transferase, whereas calf thymus alpha DNA polymerase, E. coli DNA polymerase I (Klenow fragment) and MLV reverse transcriptase do not use the nucleotide analogues as chain terminator substrates.
 ...
PMID:[Synthesis of 3'-mercapto-2',3'-dideoxynucleoside-5'-triphosphates and their properties in DNA synthesis with RNA-dependent DNA polymerases]. 768 79

N-Substituted indan-1.3-diones have proven to be potent cytotoxic agents effective against the growth of single cell leukemia tumors and cell lines derived from solid tumors. A number of the derivatives were active against growth of solid tumors e.g. colon, lung bronchogenic and osteosarcoma for which few effective agents are available to inhibit their growth. These agents inhibited DNA and RNA synthesis of L1210 cells. The de novo purine synthetic pathway was inhibited at PRPP amido transferase and IMP dehydrogenase. The pyrimidine synthetic pathway was inhibited at aspartate transcarbamylase. Other sites which demonstrate minor inhibition were DNA polymerase alpha, r- and t-RNA polymerase, ribonucleoside reductase, dihydrofolate reductase, nucleoside kinases and thymidylate synthetase. In addition d(NTP) pool levels were reduced by the drugs. L1210 DNA strand scission was evident after exposure to drugs for 24 hr. at 100 microM.
 ...
PMID:Cytotoxicity and mode of action of substituted indan-1, 3-diones in murine and human tissue cultured cells. 784 49

In the eukaryotic cell, DNA synthesis is initiated by DNA primase associated with DNA polymerase alpha. The eukaryotic primase is composed of two subunits, p49 and p58, where the p49 subunit contains the catalytic active site. Mutagenesis of the cDNA for the p49 subunit was initiated to demonstrate a functional correlation of conserved residues among the eukaryotic primases and DNA polymerases. Fourteen invariant charged residues in the smaller catalytic mouse primase subunit, p49, were changed to alanine. These mutant proteins were expressed, purified, and enzymatically characterized for primer synthesis. Analyses of the mutant proteins indicate that residues 104-111 are most critical for primer synthesis and form part of the active site. Alanine substitution in residues Glu105, Asp109, and Asp111 produced protein with no detectable activity in direct primase assays, indicating that these residues may form part of a conserved carboxylic triad also observed in the active sites of DNA polymerases and reverse transcriptases. All other mutant proteins showed a dramatic decrease in catalysis, while mutation of two residues, Arg162 and Arg163, caused an increase in Km(NTP). Analysis of these mutant proteins in specific assays designed to separately investigate dinucleotide formation (initiation) and elongation of primer indicates that these two activities utilize the same active site within the p49 subunit. Finally, mutations in three active site codons produced protein with reduced affinity with the p58 subunit, suggesting that p58 may interact directly with active site residues.
 ...
PMID:Active site mapping of the catalytic mouse primase subunit by alanine scanning mutagenesis. 787 36

Cyclic imides such as N-substituted alkyl ethers, thioethers, sulfoxides, sulfones and related derivatives were potent agents against human single cell tumors and selected solid tumor growths, eg adenocarcinoma of the colon and glioma. These agents in the L1210 lymphoid leukemia tumor model preferentially inhibited DNA synthesis. The regulatory enzyme sites in the purine pathway were targets of the agents. Other sites of inhibition were DNA polymerase alpha and thymidylate synthetase activities. d(NTP) pool levels were also reduced by the agents over 60 min.
 ...
PMID:The cytotoxic activity of cyclic imido alkyl ethers, thioethers, sulfoxides, sulfones and related derivatives. 818 34

N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines demonstrated significant cytotoxic activity against the growth of murine and human cells. These derivatives were active against leukemias, carcinomas and sarcomas. Different derivatives with N-substitutions showed specific activity against the growth of several tumor types. These agents inhibited L1210 leukemia IMP dehydrogenase and PRPP amido transferase activities; this was reflected in the inhibition of purine and DNA synthesis. Other sites inhibited to a minor degree by these agents included DNA polymerase alpha, r- and tRNA polymerases, ribonucleoside reductase, dihydrofolate reductase, pyrimidine synthesis, and nucleoside kinase. d(NTP) pool levels were reduced after 24 h incubation with these derivatives. L1210 DNA strand scission was evident after drug treatment.
 ...
PMID:The cytotoxicity of N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines. 829 66

Heterocyclic thiosemicarbazones, thioureas and 2-substituted pyridine N-oxides as well as representative nickel, cobalt and copper complexes were shown to be potent antineoplastic/cytotoxic agents. The cytotoxicity was demonstrated against single cell leukemia as well as cell lines derived from solid tissue (colon adenocarcinoma, HeLa, KB, skin, bronchogenic lung, bone osteosarcoma and glioma). In L1210 cells, DNA synthesis and subsequently RNA synthesis were particularly inhibited by the agents. IMP dehydrogenase activity and thus purine de novo synthesis was reduced significantly by the agents. Dihydrofolate reductase, ribonucleoside reductase, nucleoside kinase and DNA polymerase alpha activities were inhibited by the agents. d(NTP) pool levels were reduced by most of the agents. DNA strand scission was present with all of the derivatives; however, there was no evidence of intercalation, cross linking or alkylation/binding to bases of DNA. This new group of compounds may offer novel exploratory derivatives for future investigations in the treatment of cancer.
 ...
PMID:The cytotoxicity of heterocyclic thiosemicarbazones and their metal complexes on human and murine tissue culture cells. 849 Feb 2

A series of cyano- and carboxyborane adducts of cyclohexylamines and toluidines were shown to be cytotoxic towards suspended single cell tumors. The carboxyborane adducts of cyclohexylamine were more potent than the cyanoborane adducts of cyclohexylamine or any of the toluidine derivatives. A number of the compounds were active at 8 mg/kg/day i.p. in the Ehrlich ascites carcinoma screen in vivo. The mode of action study with N-methylcyclohexylaminecyanoborane 10 in L-1210 lymphoid leukemia cells showed that RNA synthesis was markedly reduced followed by DNA synthesis. Purine de novo synthesis was suppressed at PRPP-amido transferase, IMP dehydrogenase, and dihydrofolate reductase enzyme sites. The agent also interfered with DNA template activity causing reduction of DNA polymerase alpha, and RNA polymerase I, II and III activities. The d[NTP] pools were marginally reduced while DNA viscosity was reduced and DNA fragmentation occurred.
 ...
PMID:Synthesis and cytotoxicity of amine-borane adducts of cyclohexylamines and toluidines. 858 54


<< Previous 1 2 3 4 Next >>