Showing papers on "DNA clamp published in 1976"
TL;DR: Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase by purified from Escherichia coli cells, and the final superhelix density of the DNA can be considerably greater than that found in intracellularly super coiled DNA.
Abstract: Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.
1,108 citations
TL;DR: It is concluded that DNA gyrase controls the supercoiling of DNA in E. coli.
Abstract: Novobiocin and coumermycin are known to inhibit the replication of DNA iing of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA. The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs. The inhibition by novobiocin of colicin E1 plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase. Both in the case of coliclls. DNA molecules which are converted to the covalently circular form in thepresence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation. We conclude that DNA gyrase controls the supercoiling of DNA in E. coli.
669 citations
TL;DR: The R-loop formation is maximal at the temperature at which half of the duplex DNA is irreversibly converted to single-stranded DNA and falls precipitously a few degrees above or below that temperature and the rate is proportional to the RNA concentration.
Abstract: RNA can hybridize to double-stranded DNA in the presence of 70% formamide by displacing the identical DNA strand. The resulting structure, called an R-loop, is formed in formamide probably because of the greater thermodynamic stability of the RNA-DNA hybrid when it is near the denaturation temperature of duplex DNA. The rate of R-loop formation is maximal at the temperature at which half of the duplex DNA is irreversibly converted to single-stranded DNA (the strand separation temperature of tss) of the duplex DNA and falls precipitously a few degrees above or below that temperature. This maximal rate is similar to the rate of hybridization of RNA to single-stranded DNA under the same conditions. At temperatures above the tss the rate is proportional to the RNA concentration. However, at temperatures below tss the rate of R-loop formation is less dependent upon the RNA concentration. Once formed, the R-loops display considerable stability; the formamide can be removed and the DNA can be cleaved with restriction endonucleases without loss of R-loop structures.
367 citations
TL;DR: Endonucleases that specifically attack DNA containing pyrimidine dinners2 or apurinic sites have been isolated from many types of cells, and enzymes of these two types have been shown to be active in DNA repair in Escherichia coli by the isolation of repair-deficient mutant strains with defective endo-nucleases.
Abstract: ONE of the major pathways of DNA repair consists of the removal and replacement of damaged nucleotides in non-replicating DNA. In the classical excision repair model1,2, the first enzymatic step in this process is the introduction of a single-strand break in the DNA adjacent to a defective nucleotide residue. Endonucleases that specifically attack DNA containing pyrimidine dinners2 or apurinic sites3–5 have subsequently been isolated from many types of cells, and enzymes of these two types have been shown to be active in DNA repair in Escherichia coli by the isolation of repair-deficient mutant strains with defective endo-nucleases6,7.
247 citations
TL;DR: A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow and results are consistent with the coexistence of two enzyme activities in a single protein.
Abstract: A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow. This DNA polymerase, in contrast to previously described eukaryotic DNA polymerases, is associated with a very active 3' to 5' exonuclease activity. Similar to the 3' to 5' exonuclease activity associated with prokaryotic DNA polymerases, this enzyme catalyzes the removal of 3'-terminal nucleotides from DNA, as well as a template-dependent conversion of deoxyribonucleoside triphosphates to monophosphates. The exonuclease activity is not separable from the DNA polymerase activity by chromatography on DEAE-Sephadex or hydroxylapatite, and upon sucrose density gradient centrifugation the two activities cosediment at 7 S or at 11 S depending on the ionic strength. Both exonuclease and polymerase activities have identical rates of heat inactivation and both are equally sensitive to hemin and Rifamycin AF/013, inhibitors of DNA synthesis that act by binding to DNA polymerase and causing its dissociation from its template/primer. These results are consistent with the coexistence of two enzyme activities in a single protein.
233 citations
TL;DR: Production of protein X is part of a feedback loop that limits DNA degradation and allows DNA repair, and is suggested to be an indirect consequence of DNA synthesis inhibition.
231 citations
TL;DR: Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out and demonstrated that phosphono acetate inhibited the polymerase by interacting with it at the pyroph phosphate binding site to create an alternate reaction pathway.
Abstract: Phosphonoacetate was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out The results demonstrated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway A detailed mechanism and rate equation for the inhibition were developed For comparison to phosphonoacetate, pyrophosphate inhibition patterns and apparent inhibition constants were determined Twelve analogues of phosphonoacetate were tested as inhibitors of the herpesvirus of turkey-induced DNA polymerase At the concentrations tested, only one, 2-phosphonopropionate, was an inhibitor The apparent inhibition constant for it was about 50 times greater than the corresponding apparent inhibition constant for phosphonoacetate DNA polymerase alpha of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phosphonoacetate The apparent inhibition constants for the alpha polymerase were about 10-20 times greater than the corresponding inhibition constants for the herpesvirus-induced DNA polymerase Duck DNA polymerase beta, Escherichia coli DNA polymerase I, and avian myeloblastosis virus reverse transcriptase were not inhibited by phosphonoacetate
206 citations
TL;DR: An analysis of the replicating molecules in sucrose density gradients reveals that there are AAV DNA intermediates which consist of covalently linked plus and minus DNA strands, compatible with a self-priming mechanism for the synthesis of AAVDNA.
Abstract: Replicating DNA molecules of adenovirus-associated virus (AAV) were selectively extracted from KB cells coinfected at 395 detrees with a DNA minus, temperature-sensitive mutant of adenovirus 5 (ts125) as helper Under these conditions AAV DNA replication proceeds normally, but there is little, if any, adenovirus DNA synthesis An analysis of the replicating molecules in sucrose density gradients reveals that there are AAV DNA intermediates which consist of covalently linked plus and minus DNA strands Under denaturing conditions, these concatemers are linear single strands whose lengths can reach at least four times the size of the AAV genome The most abundant concatemeric species is a dimer which presumably exists in vivo as a unit length hairpin Unit length linear duplexes appear to be immediate precursors of plus and minus progeny strands These findings are compatible with a self-priming mechanism for the synthesis of AAV DNA
196 citations
TL;DR: Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.
Abstract: DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.
127 citations
TL;DR: It is concluded that a function of thePolC gene is necessary for error-prone repair to occur and that this function is defective at 43° in the enzyme specified by the polC allele from BT1026.
Abstract: The polC (= dnaE) temperature-sensitive DNA polymerase III mutation from Escherichia coli BT1026 has been transduced into E. coli WP2 (to give CM731) and WP2 uvr A (to give CM741). In excision-deficient CM741 UV-induced Trp+ mutations progressively lost their photoreversibility during post-irradiation incubation at 34 degrees. Immediately after transfer to 43 degrees, however, there was no further loss of reversibility although post-replication strand joining still occurred and uptake of 3H-thymidine into DNA continued for 20 to 30 min. In excision-proficient CM731, UV lesions capable of leading to Strr mutations disappeared during post-irradiation incubation at restrictive temperature and there was no increase in the number remaining after exposure to photoreversing light. In contrast, at permissive temperature, premutational lesions were not lost and became progressively converted into non-photoreverisble mutations. It is concluded that a function of the polC gene is necessary for error-prone repair to occur and that this function is defective at 43 degrees in the enzyme specified by the polC allele from BT1026. This function seems not to be essential for most post-replication or excision repair or for normal DNA replication and may be particularly involved in the insertion of incorrect bases during error-prone repair.
122 citations
TL;DR: DNA polymerase activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared and it was observed that the enzyme from late passage cells was more error-prone.
Abstract: DNA polymerase (deoxynucleosidetriphosphate: DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.
TL;DR: Elongation of a primed single-stranded DNA template catalyzed by E. coli DNA polymerase III requires dnaZ protein and two other protein factors, DNA elongation factors I and III, and catalyzes DNA synthesis upon the addition of dNTPs.
Abstract: Elongation of a primed single-stranded DNA template catalyzed by E coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2777) requires dnaZ protein and two other protein factors, DNA elongation factors I and III The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs
TL;DR: The results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted.
Abstract: The genome of the nondefective parvovirus minute virus of mice (MVM) is a linear DNA molecular weight 1.48 x 10(6), which is single stranded for approximately 94% of its length. In contrast to the genomes from defective parvoviruses MVM DNA does not contain a detectable inverted terminal redundancy. A combination of enzymatic and physical techniques has shown that the molecule contains a stable hairpin duplex of approximately 130 base pairs located at the 5' terminus of the genome. MVM DNA is efficiently utilized as a template-primer by a number of DNA polymerases, including reverse transcriptases. Polymerases lacking 5' to 3' exonuclease activity yield a duplex DNA product with a molecular weight 1.96 times that of the viral genome, in which the newly synthesized complementary strand is covalently attached to the template. This duplex contains an internal "nick" that can be sealed by DNA ligase to produce a self-complementary single-strand circle. The MVM DNA duplex is cleaved twice by EcoR-RI restriction endonuclease to yield three distinct fragments in molar amounts. These results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the 3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted.
TL;DR: Two further points of analogy are demonstrated: both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation.
TL;DR: The weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA.
Abstract: The effect of several divalent cations on the accuracy of DNA replication in vitro has been examined. Only Be2+ altered the accuracy of DNA synthesis using purified DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) from avian myeloblastosis virus. The Be2+-induced base substitutions occurred with all templates and with all nucleotides tested. Analysis of the product by equilibrium density centrifugation and processive hydrolysis with snake venom phosphodiesterase suggested that the noncomplementary nucleotides were present in phosphodiester linkage. Nearest neighbor studies indicated that many of the Be2+-induced errors were present as single base substitutions. The enhancement of error frequency could be duplicated by the pretreatment of the enzyme, but not the template, with Be2+. Glycerol gradient centrifugation dissociated the Be2+-DNA polymerase complex and restored the initial error frequency of the polymerase. Thus, the weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA. Beryllium is a known carcinogen. The potential use of this system as a screening technique to detect chemical mutagens and carcinogens is considered.
TL;DR: The results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it, and by measuring maximum levels of methylation of DNAs from different sources the proportion of unmethylated sites present in them is estimated.
Abstract: DNA methylase has been purified 405-fold from Krebs II ascites cells. The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine. Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations. Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it. By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them. Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA. DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.
TL;DR: The levels of the DNA polymerases-alpha and -beta in phytohemagglutinin-stimulated lymphocytes during a 12-day period suggest a certain tendency to the specialization of functions in human cell DNA polymerase.
Abstract: Stimulation of human lymphocytes with phytohemagglutinin is known to induce an increase in overall DNA polymerase activity (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7). Previous work [Pedrali Noy, G., Dalpra, L. Pedrini, A. M., Ciarrocchi, G., Giulotto, E., Nuzzo, F. & Falaschi, A. (1974) Nucleic Acids Res. 1, 1183] has shown that two subsequent waves of induction of DNA polymerase can be observed in this system; a first wave occurs in parallel with the increase in DNA replication rate; a second one occurs when the DNA synthesis rate is returned to minimal levels; the second peak is parallel to a maximum in DNA ligase and DNase levels. In the present work we have measured the levels of the DNA polymerases-alpha and -beta in phytohemagglutinin-stimulated lymphocytes during a 12-day period; both enzymes are present at detectable levels at time zero; in correspondence to the peak of DNA synthesis rate (between the fourth and fifth day) a peak of DNA polymerase-alpha is observed, increasing by a factor of approximately 20-fold over the zero time value; subsequently, the level of DNA polymerase-alpha decreases in parallel with DNA synthesis rate. The DNA polymerase-beta is also increased in correspondence to the peak in DNA synthesis rate, but reaches its maximum at later times, between the eighth and tenth day of incubation. The capacity of stimulated lymphocytes to perform repair synthesis following UV damage was measured in the same cells used for the enzyme activity determinations; this capacity also shows two maxima: a first one correlated with the peak in DNA replication rate, and a second one correlated with the peak of DNA polymerase-beta. These data suggest a certain tendency to the specialization of functions in human cell DNA polymerases; the alpha-enzyme seems mainly correlated with DNA replication, whereas the beta-enzyme seems more correlated with the ability of the cell to perform repair type synthesis.
TL;DR: Homogeneous DNA polymerase from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity and failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.
TL;DR: It is determined that Co2+, Ni2+ or Zn2+ may substitute for Mg2+ during DNA synthesis with E.coliDNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerasing virus (AMV).
TL;DR: DNA polymerase I has been purified to greater than 90% homogeneity from a strain of Escherichia coli K12 that bears the temperature-sensitive DNA polymerases I mutatation, polA12, and its capacity to promote the concerted 5' leads to 3' polymerization is decreased 10-fold, probably the result of a significant alteration in the tertiary structure of the enzyme.
TL;DR: The idea that α-type polymerases are involved in the replicative synthesis of cellular and viral DNA is supported, as HCMV infection induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA.
TL;DR: It is shown that the CB120 antimutator polymerase also initially selects incorrect nucleotides for incorporation less frequently than the wild type enzyme, and appears to have both greater accuracy in nucleotide selection and an enhanced ability to remove incorrectucleotides.
TL;DR: It is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.
TL;DR: A separate stage in strand initiation is identified which precedes dnaG RNA polymerase participation and appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.
Abstract: Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.
TL;DR: In this paper, temperature-sensitive Escherichia coli cell division mutants and DNA repair mutants were treated in several ways to alter DNA synthesis or cell division, and autoradiography was performed on the slab gels so that the rate of synthesis of protein X could be determined by micro-densitometry.
TL;DR: DNA polymerase III from Bacillus subtilis has been purified about 4,500-fold and reveals a single major protein band which co-migrates with the polymerase activity and appears to contain a single, long, polypeptide chain.
TL;DR: Using a membrane filter assay, results are obtained indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA.
Abstract: Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.
TL;DR: Electron microscopic examination of intracellular viral DNA purified by equilibrium sedimentation in CsCl revealed DNA “tangles,” as well as longer than unit-size linear molecules, which indicate that concatemeric forms of viral DNA are present in the infected cells.
TL;DR: Data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism, and some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template.
Abstract: Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.
TL;DR: The data suggests a “brush-like” model for the intracellular T4 chromosome, with the DNA held at roughly genome-length intervals by some non-DNA core material resistant to both strong detergent and protease treatments.