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)

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.
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PMID:The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA. 610 97

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.
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PMID:Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA. 611 28

Five chromatographically distinct DNA-dependent ATPase activities have been identified in high salt-detergent extracts of the Novikoff hepatoma. One of these, ATPase III, has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis and has a specific activity of 12 mumol of ATP hydrolyzed min-1 (mg of protein)-1. The enzyme, a dimer of Mr 65000 subunits, has a sedimentation coefficient of 7.0 S in both high salt and low salt, a Stokes radius of 43 A, and a frictional coefficient of 1.31. In the presence of Mg2+ ion and a polynucleotide effector, the enzyme catalyzes hydrolysis of ATP or dATP to a diphosphate with a Km of 206 microM and 110 microM, respectively, for the two substrates. Although single-stranded effectors are preferred, the enzyme has significant activity with double-stranded effectors. The Km for effector is 0.4 microM (nucleotide). The analogues adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), dideoxyadenosine triphosphate (ddATP), and adenosine 5'-(alpha, beta-methylenetriphosphate) (alpha, beta-Me-ATP) are competitive inhibitors of the enzyme while adenosine tetraphosphate (ATP-P), 8-bromoadenosine 5'-triphosphate (8-Br-ATP), 5'-adenylyl imidodiphosphate (AMP-PNP), and adenosine 5'-(beta, gamma-methylenetriphosphate) (beta, gamma-Me-ATP) do not inhibit. The enzyme is insensitive to nalidixic acid, novobiocin, and berenil but is sensitive to N-ethylmaleimide. ATPase III is capable of stimulating DNA polymerase beta on duplex DNA, but this effect is abolished in the presence of ATP gamma S. Polymerase stimulation is further enhanced in the presence of a single-stranded DNA-binding protein. These data suggest that ATPase III may play a role in DNA repair.
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PMID:Deoxyribonucleic acid dependent adenosinetriphosphatases from the Novikoff hepatoma. Characterization of a homogeneous adenosinetriphosphatase that stimulates DNA polymerase beta. 612 27

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.
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PMID:DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7. 613 75

A new DNA-dependent ATPase named ATPase IV has been purified to apparent homogeneity from Escherichia coli as a by-product of DNA polymerase III purification. The enzyme has a specific activity of 360 mumol of ATP hydrolyzed per min/mg of protein. The purified enzyme exists as monomer with a molecular weight of 81,000. It sediments in a glycerol gradient as a single species of 4.5 S. The enzyme has considerable activity at 0 degree C and has a Q10 of 3.8. In the presence of a DNA effector and magnesium ion, the enzyme will hydrolyze ATP, dATP, GTP, or dGTP to a nucleoside diphosphate plus orthophosphate with a Km of 0.20, 0.50, 0.60, and 1.30 mM, respectively. The guanine nucleotides, however, are only 25-35% as effective as substrates compared with the adenine nucleotides. ATPase IV shows strong substrate inhibition by ATP, but not dATP, above 0.2 mM. The polynucleotide effector requirement can be satisfied by either single-stranded or double-stranded DNA. The enzyme binds the effector very tightly with a Km of 3 X 10(-8) M (nucleotide) for G4 DNA. The enzyme is inhibited by E. coli single-stranded DNA-binding protein, a variety of ATP analogues and N-ethylmaleimide. The relationship of ATPase IV to DNA polymerase III holoenzyme is discussed.
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PMID:A new DNA-dependent ATPase from Escherichia coli. Purification and characterization of ATPase IV. 614 53

Protein factors have been isolated from HeLa cells and from calf thymus which are able to specifically stimulate DNA polymerase alpha in vitro on templates which mimic the replication fork. One factor, extracted from HeLa cells, is an enzymatic complex of about 100-110 Kdal composed of a DNA-dependent ATPase and of an as yet uncharacterized DNA-binding protein. This complex exhibits a limited "helicase" activity on DNA : DNA partial duplexes which probably accounts for the stimulation of DNA polymerase alpha. The other stimulatory factor is obtained from calf thymus. They are the so-called single-stranded DNA binding proteins (DBP) which have a duplex-destabilizing activity. These proteins appear to be heterogeneous with regard to both physical properties (Mr and pI) and functional characteristics (DNA polymerase alpha stimulation, duplex denaturation). The origin and the biological significance of the different molecular forms are discussed.
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PMID:DNA polymerase accessory proteins in mammalian cells. 614 84

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.
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PMID:The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates. 623 26

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and beta-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H(3) (32)PO(4) were shown to utilize internally generated adenosine 5'-triphosphate in the phosphorylation of amikacin. The absence of (32)P-phosphorylated amikacin after incubation of [gamma-(32)P]adenosine 5'-triphosphate with spheroplasts confirmed that exogenous adenosine 5'-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.
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PMID:Localization of an amikacin 3'-phosphotransferase in Escherichia coli. 626 7

A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.
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PMID:Stimulation of DNA polymerase alpha by a nuclear DNA/protein complex. 627 80

The limitations to SV40 growth in nonpermissive cells are poorly understood. In differentiated mouse cells, early mRNA and T-antigens are synthesized, but no viral DNA replication has been detected. A plausible explanation for the limitation to viral DNA synthesis in these cells might be the inability of a mouse cell-specific DNA polymerase to interact with the SV40 T-antigen-viral DNA complex. In spite of this abortive viral-cell interaction, SV40 late viral transcripts can be detected in infected mouse cells. Both early and late transcripts can be detected in infected mouse cells. Both early and late SV40 transcriptional activities peak between 10 and 15 h post infection; by 24 h after infection SV40 RNA is almost undetectable. In spite of the detection of late SV40 mRNA in mouse cells, we have been unable to detect any structural proteins (VP1, VP2, or VP3) encoded by these transcripts. A more restrictive interaction occurs between SV40 and undifferentiated mouse teratocarcinoma cell lines. Using an in vitro technique, the viral transcriptional complex (VTC) assay, we were able to demonstrate viral transcriptional activity on both the early and the late strands of SV40 in F9 embryonal carcinoma cells. Nevertheless, no mature processed mRNAs or viral encoded polypeptides were detected in these cells. After differentiation of the F9 cells with retinoic acid, however, spliced early mRNAs, as well as the SV40 T-antigen, were present.
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PMID:The regulation of SV40 gene expression in nonpermissive cells. 627 31


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