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)

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.
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PMID:Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA. 12 75

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.
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PMID:A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication. 13 39

Phage fd DNA complexed with DNA binding protein I was used by Escherichia coli RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) to synthesize an RNA at the origin of single strand to double strand replication. The isolated ori-RNA gave a simple fingerprint after nucleolytic digestion and has a length of about 30 nucleotides. The characterization of the oligonucleotides from the nuclease digest and the extension of the ori-RNA with DNA polymerase I and subsequent restriction of the DNA gave its exact localization in the fd genome, and its total sequence was deduced from the known DNA sequence in this region.
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PMID:An RNA transcribed from DNA at the origin of phage fd single strand to replicative form conversion. 20 27

During the productive infection of KB cells by adenovirus type 5 (Ad5), there is a progressive decrease in the level of cellular DNase activity towards single-stranded DNA, in contrast to DNA polymerase which remains relatively constant throughout the infection. This decrease is prevented by the inhibition of protein synthesis by cycloheximide. The inhibition of DNase activity does not occur after infection by Ad5 ts125, a DNA-negative mutant which fails to induce the adenovirus-specific DNA binding protein. In contrast, infection by Ad5 ts36, a DNA-negative mutant which complements ts125, does result in decreased levels of DNase. A mechanism is discussed in which the DNA binding protein protects viral replicative intermediates from degradation by cellular DNase.
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PMID:Adenovirus-induced inhibition of cellular DNase. 20 1

DNA polymerase and gene 4 protein of bacteriophage T7 catalyze DNA synthesis on duplex DNA templates. Synthesis is initiated at nicks in the DNA template, and this leading strand synthesis results in displacement of one of the parental strands. In the presence of ribonucleoside 5'-triphosphates the gene 4 protein catalyzes the synthesis of oligoribonucleotide primers on the displaced single strand, and their extension by T7 dna polymerase accounts for lagging strand synthesis. Since all the oligoribonucleotide primers bear adenosine 5'-triphosphate residues at their 5' termini, [gamma 32P]ATP is incorporated specifically into the product molecule, thus providing a rapid and sensitive assay for the synthesis of the RNA primers. Both primer synthesis and DNA synthesis are stimulated 3- to 5-fold by the presence of either Escherichia coli or T7 helix-destabilizing protein (DNA binding protein). ATP and CTP together fully satisfy the requirement for rNTPs and provide maximum synthesis of primers and DNA. Provided that T7 DNA polymerase is present, RNA-primed DNA synthesis occurs on either duplex or single-stranded DNA templates and to equal extents on either strand of T7 DNA. No primer-directed DNA synthesis occurs on poly(dT) or poly(dG) templates, indicating that synthesis of primers is template-directed.
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PMID:Requirements for synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and gene 4 protein of bacteriophage T7. 22 44

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.
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PMID:Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7. 22 45

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.
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PMID:Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase. 22 73

Escherichia coli dnaG protein is involved in the initiation of DNA synthesis dependent on G4 or ST-1 single-stranded phage DNAs [Bouche, J.-P., Zechel, K & Kornberg, A. (1975) J. Biol. Chem. 250, 5995-6001]. The reaction occurs by the following mechanism: dnaG protein binds to specific sites on the DNA in a reaction requiring E. coli DNA binding protein. An oligonucleotide is synthesized in a reaction involving dnaG protein, DNA binding protein, and DNA. With G4 DNA this reaction requires ADP, dTTP (or UTP), and dGTP (or GTP). Elongation of the oligonucleotide can be catalyzed by DNA polymerase II or III in combination with dnaZ protein and DNA elongation factors I and III, presumably by the mechanism previously reported [Wickner, S. (1976) Proc. Natl. Acad. Sci. USA 73, 3511-3515] or by DNA polymerase I.
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PMID:DNA or RNA priming of bacteriophage G4 DNA synthesis by Escherichia coli dnaG protein. 26 32

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.
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PMID:phiX174 cistron A protein is a multifunctional enzyme in DNA replication. 26 83

Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome. Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription. The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together. Both RNA and DNA primers were initiated with ATP. At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers. Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme. At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates. Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues. The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template. The multienzyme priming system is highly specific for phiX174 DNA as template.
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PMID:A multienzyme system for priming the replication of phiX174 viral DNA. 34 90


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