Showing papers on "Primase published in 1974"
TL;DR: Understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.
Abstract: Replication is accomplished by multienzyme systems whose operations are usefully considered in respect to three stages of the process: initiation, elongation, anid termination. 1) Initiation entails synthesis of a short RNA fragment that serves as primer for the elongation step of DNA synthesis. This stage, probed by SS phage DNA templates, reveals three distinctive and highly specific systems in E. coli. The Ml3 DNA utilizes RNA polymerase in a manner that may reflect how plasmid elements are replicated in the cell. The OX174 DNA does not rely on RNA-polymerase, but requires instead five distinctive proteins which may belong to an apparatus for initiating a host chromosome replication cycle at the origin. The G4 DNA, also independent of RNA polymerase, needs simply the dnaG protein for its distinctive initiation and may thus resemble the system that initiates the replication fragments at the nascent growing fork. In each case it is essential that in vitro the DNA-unwinding protein coat the viral DNA and influence its structure. 2) Elongation is achieved in every case by the multisubunit, holoenzyme form of DNA polymerase III. Copolymerase III, which is an enzyme subunit, and adenosine triphosphate are required to form a proper complex with the primer template but appear dispensable for the ensuing chain growth by DNA polymerase (33). 3) Termination requires excision of the RNA priming fragment, filling of gaps and sealing of interruptions to produce a covalently intact phosphodiester backbone. DNA polymerase I has the capacity for excision and gapfilling and DNA ligase is required for sealing. What once appeared to be a simple DNA polymerase-mediated conversion of a single-strand to a duplex circle (34) is now seen as a complex series of events in which diverse multienzyme systems function. Annoyance with the difficulties in resolving and reconstituting these systems is tempered by the conviction that these are the very systems used ,by the cell in replicating its chromosome and extrachromosomal elements. Thus, understanding of the regulation of replication events in the cell, their localization at membrane surfaces and integration with cell division, and their coordination with phage DNA maturation and particle assembly will all be advanced by knowledge of the components of the replicative machinery.
191 citations
TL;DR: The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex DNA have now been purified extensively and DNA synthesis by these proteins requires varphi X174 viral DNA, dNTPs, Mg(+2), and ATP.
Abstract: The E. coli proteins that catalyze the conversion of ϕX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires ϕX174 viral DNA, dNTPs, Mg+2, and ATP. The product synthesized is full-length linear ϕX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and ϕX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.
152 citations
TL;DR: The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs, which supports the hypothesis that each of the 35S RNA subunits of M- Mu LV 60- 70S RNA has a different base sequence.
Abstract: The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.
144 citations
TL;DR: A possible physiological role for DNA polymerase II and the unwinding protein of E. coli is suggested to be essential for the synthesis of a DNA product that is equal in size to the template.
108 citations
TL;DR: The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus, and these primers were purified by isolation of the covalent complex between primer and nascent DNA, differential melting of the 70SRNA, and by two-dimensional electrophoresis in polyacrylamide gels.
Abstract: The 70S RNA of Rous sarcoma virus contains 4S RNAs which serve as primers for the initiation of DNA synthesis in vitro by the RNA-directed DNA polymerase of the virus. We purified these primers in three different ways—by isolation of the covalent complex between primer and nascent DNA, by differential melting of the 70S RNA, and by two-dimensional electrophoresis in polyacrylamide gels. The 4S RNAs purified by these procedures were homogeneous and possessed very similar if not identical nucleotide compositions and sequences. The RNAs were approximately 75 nucleotides long, had pG at the 5′ terminus and CpCpAOH at the 3′ terminus, and contained a number of minor nucleotides characteristic of tRNA. In contrast to most tRNA's, the primer lacked rTp and contained Gp (Ψp, Ψp, Cp) Gp (possibly in place of the characteristic sequence GprTpΨpCpGp). At least 50% of the 4S primers available on 70S RNA were utilized in a standard polymerase reaction in vitro.
88 citations
TL;DR: T4 DNA polymerase cannot use a nicked duplex DNA molecule as a template-primer for DNA synthesis, apparently because it is unable to displace the 5' end of the strand paired to the strand serving as the template.
88 citations
TL;DR: Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit, which is a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.
85 citations
TL;DR: A technique for isolating and separating DNA polymerases in mammalian cells infected with an RNA tumor virus is described and designed for further application to the study of DNA polymerase in human leukemic cells and other human tumors.
70 citations
TL;DR: During active growth of cells polymerase activity increases about 1.4-fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A, which together with other properties suggests a role of this enzyme in DNA replication.
Abstract: Yeast DNA polymerases A and B were purified 5000 – 10000-fold by chromatography on DEAE-cellulose, phosphocellulose, DNA-agarose and DEAE-Sephadex. In acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, both enzymes give rise to three main bands. The enzymes are equally susceptible to inhibition by the – SH reagents N-ethylmaleimide and p-chloromercuri-benzoate but differ in their sensitivity to cytosine-arabinoside triphosphate, polymerase A being considerably more sensitive to this nucleotide analog. Whereas DNA polymerase A prefers nicked DNA and poly[d(A-T)] as template-primer, polymerase B is most active with poly(dA) · (dT)10. Km values for deoxyribonucleoside triphosphates were determined as 3.7–3.9 μM for enzyme A and 1.8–2.4 μM for enzyme B. During active growth of cells polymerase activity increases about 1.4-fold over the amount found in resting cells, which is mainly if not exclusively due to an increased level of DNA polymerase A. This together with other properties suggests a role of this enzyme in DNA replication.
63 citations
TL;DR: These two factors are also essential for dTMP incorporation catalyzed by DNA polymerase III and dependent on poly(dA).oligo(dT) primer-template in the latter reaction, there is an absolute dependency on ATP or dATP.
Abstract: The in vitro conversion of single-stranded DNA from bacteriophage fd to duplex structures depends on E. coli RNA polymerase, DNA polymerase III, riboand deoxyribonucleoside triphosphates, Mg+2, spermidine or DNA-unwinding protein of E. coli, and two additional protein factors, referred to here as Factors I and II. These two factors are also essential for dTMP incorporation catalyzed by DNA polymerase III and dependent on poly(dA)·oligo(dT) primer-template. In the latter reaction, there is an absolute dependency on ATP or dATP.
56 citations
TL;DR: The 5'→3' exonuclease can excise thymine dimers from DNA in vitro, and there is genetic evidence that DNA polymerase I play a role in repair of damaged DNA in vivo.
Abstract: Publisher Summary DNA polymerase I is a single polypeptide chain of molecular weight 109,000, and the absorption of a 1 mg/ml solution is 085 at 278 nm The enzyme contains two molecules of tightly bound zinc that may be involved in enzyme activity and single disulfide and sulfhydryl groups Polymerase I from Escherichia coli is a multifunctional enzyme catalyzing three reactions in vitro that may be important in vivo In addition to phosphodiester bond formation (DNA synthesis), the enzyme also catalyzes two distinct exonucleolytic reactions: one degrading DNA in the 3'→5' direction (3'→5' exonuclease), and the other degrading DNA in the 5'→3' direction (5'→3' exonuclease) The 5'→3' exonuclease can excise thymine dimers from DNA in vitro , and there is genetic evidence that DNA polymerase I play a role in repair of damaged DNA in vivo
TL;DR: Short fragments of DNA isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria, compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer.
Journal Article•
TL;DR: The suggestion that polymerase I is involved in DNA replication is consistent with the finding that, in the presence of “activated” DNA, it is inhibited to a greater extent by ara-CTP than is polymerase II.
Abstract: The Km for dCTP and the K1 for 1-β-d-arabinofuranosylcytosine 5′-triphosphate (ara-CTP) were determined with DNA polymerases purified by diethylaminoethyl cellulose and phosphocellulose chromatography from Rauscher murine leukemia virus, from human blood lymphocytes, and from a human lymphoblastoid cell line. Inhibition of the viral reverse transcriptase varied with the template/primer used. With poly(rI)·oligo(dC) (rIn·dC12-18) as the synthetic template/primer, this enzyme had a greater relative affinity for the inhibitor than had the cellular enzymes in the presence of DNA. However, when DNA was the template, inhibition of the viral enzyme was decreased considerably. DNA polymerase I of normal human lymphoid cells, which was separated from the lowermolecular-weight polymerase II and could be assayed only in the presence of DNA, had a lower relative affinity for ara-CTP than the viral reverse transcriptase directed by poly(rI)·oligo(dC). However, in the presence of magnesium ions and a DNA template, the viral enzyme was inhibited far less than either of the cellular enzymes. Since ara-CTP inhibits both viral and cellular DNA polymerases and inhibition varies with the template used, the compound cannot be considered a specific inhibitor of reverse transcriptase in studies of virus-cell interactions. The suggestion that polymerase I is involved in DNA replication is consistent with the finding that, in the presence of “activated” DNA, it is inhibited to a greater extent by ara-CTP than is polymerase II.
TL;DR: Bleomycin inhibits vaccinia virus replication in infected HeLa cells and in mice and effects on DNA synthesis, in particular, the induced degradation of template DNA may be responsible for the observed antiviral activity of bleomycin.
TL;DR: The addition of 10% glycerol and/or 2 mM spermidine to extracts blocks synthesis of completely replicated molecules while enhancing synthesis of molecules containing newly synthesized DNA fragments.
Abstract: The major products of colicin E1 plasmid DNA synthesis in cell extracts are completely replicated molecules and a class of molecules containing newly synthesized small DNA fragments. The addition of 10% glycerol and/or 2 mM spermidine to extracts blocks synthesis of completely replicated molecules while enhancing synthesis of molecules containing newly synthesized DNA fragments. The latter molecules, which contain on the average two DNA fragments of approximately 6 S, are early replicative intermediates for synthesis of completely replicated molecules. Synthesis of the intermediates is sensitive to rifampicin and depends on RNA synthesis. RNA components are linked to the 6S DNA molecules.
TL;DR: The degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase : DNA ratio.
Abstract: The routine procedures used to prepare DNA from eucaryotic cells and nuclei give rise to material which is considerably degraded. This degradation takes the form of haplotomic (single-stranded) breaks arising from deoxyribonuclease action action and diplotomic cleavage (double-stranded scission) of the DNA duplex due to mechanical shearing during the isolation procedure. Attempts to study the template specificities of the eucaryotic DNA-dependent RNA polymerases must take into account how these modifications to the structure of the DNA affect its template properties for these enzymes. Techniques are described which permit an accurate assessment of the state of integrity of the DNA. Treatment of the DNA with pancreatic deoxyribonuclease or sonication is shown to give rise to single-stranded interruptions in the duplex which are competent sites for the initiation of RNA synthesis by both forms AI and B rat liver RNA polymerases. However both these treatments also give rise to diplotomic cleavage of the DNA. This results in an inhibition of RNA synthesis which may be correlated with the formation of non-productive complexes by the RNA polymerases at DNA ends. Therefore the degree of template activation by pancreatic deoxyribonuclease and sonication for RNA synthesis as seen in the assay in vitro is a delicate balance between two opposing effects and is highly dependent upon the polymerase : DNA ratio. This data serves to emphasise that experiments designed to study the template specificity of eucaryotic RNA polymerases must make use of new, sophisticated techniques for the preparation of a template which is both intact and of high molecular weight.
TL;DR: Evidence is presented which indicates that at least part of the ultraviolet protection conferred on wild-type cells by R-Utrecht can be attributed to a plasmid-coded DNA polymerase which participates in repair of single strand gaps.
Abstract: CERTAIN plasmids, including the colicin factor ColIb and the drug resistance transfer factor R-Utrecht, reduce the susceptibility of wild-type cells of Salmonella typhimurium to the lethal effects of ultraviolet irradiation while at the same time increasing their susceptibility to the mutagenic effects1,2. These alterations in ultraviolet response may result from plasmid-borne genes whose products participate in repair of single-strand gaps in DNA (ref. 2). Here I present evidence which indicates that at least part of the ultraviolet protection conferred on wild-type cells by R-Utrecht can be attributed to a plasmid-coded DNA polymerase which participates in repair of single strand gaps.
TL;DR: Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments.
Abstract: Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture.
TL;DR: This chapter deals with the recent developments concerning the isolation and characterization of DNA polymerases II and III of Escherichia coli and the properties of the first DNA polymerase to be isolated from E. coli.
Abstract: Publisher Summary This chapter deals with the recent developments concerning the isolation and characterization of DNA polymerases II and III of Escherichia coli. Genetic and physiological experiments designed to elucidate the biological function of these enzymes are also discussed. The chapter explores the properties of the first DNA polymerase to be isolated from E. coli, DNA polymerase. The isolation of a mutant of E. coli defective in DNA polymerase I activity (PolA 1 - ) provided the basis for several investigations into the nature of the enzymatic mechanism of DNA replication in such cells. After the isolation of the PolA 1 - mutant, it was generally assumed, since no other polymerase had yet been discovered in E. coli, that either residual amounts of DNA polymerase I or some yet undiscovered polymerase with perhaps unusual requirements for activity provided the enzymatic activity required for DNA replication. Initially, we described an enzyme activity in PolA 1 - cells that catalyzed the synthesis of DNA. This activity was resistant to antiserum capable of inactivating DNA polymerase I. It was also discovered that there indeed exists another DNA polymerase activity in E. coli and the isolation and purification of this enzyme, DNA polymerase II, from cells lysed after toluene or detergent treatment and by more conventional means were reported. No strong evidence yet exists that the preparation of this enzyme requires any unconventional procedures; its low activity compared to that of DNA polymerase I probably accounts for the fact that it was previously unnoticed in wild-type E. coli.
TL;DR: Ncent DNA synthesized from RNase-sensitive endogenous DNA polymerase activities of microsomal and mitochondrial fractions from phytohemagglutinin-stimulated normal human blood lymphocytes is covalently attached to a small RNA molecule.
TL;DR: The frequency with which a non-complementary base-paired nucleotide is incorporated by the DNA polymerase of Rauscher leukemia virus is determined using synthetic polynucleotides as templates, suggesting that a high level of infidelity may be a common characteristic of RNA tumor viruses.
TL;DR: Participatory roles of the three E. coli DNA polymerases in each of these stages has been determined and will help improve the understanding of how DNA replication occurs in the cell.
Abstract: Chain elongation during E. coli DNA replication proceeds in three distinct stages. Participatory roles of the three E. coli DNA polymerases in each of these stages has been determined.
TL;DR: A general approach for determining the nucleotide sequence of a gene, with the aid of a deoxyribonucleotide primer of defined sequence, and the result indicated that this primer was bound to the expected location on the T4 DNA.
Abstract: We have developed a general approach for determining the nucleotide sequence of a gene, with the aid of a deoxyribonucleotide primer of defined sequence. The selection of the primer sequence was based on a short segment of mRNA sequence of T4 phage lysozyme. A tetradecadeoxyribonucleotide primer was chemically synthesized and its sequence verified by sequence analysis. This primer was found to bind to the single-stranded region of the exonuclease III-treated T4 DNA, and specific nucleotides were incorporated to its 3′ end. The result indicated that this primer was bound to the expected location on the T4 DNA. Therefore, long sequences of the T4 lysozyme gene can now be determined from this specific starting point.
TL;DR: The investigations into the residual polymerase activity in polA — ceils have resulted in the isolation of two other DNA polymerases called polymerase II and polymerase III, in order of discovery.
Abstract: Publisher Summary Three DNA polymerases have been isolated from Bacillus subtilis. The first of these, polymerase I, was purified in 1964 by Okazaki and Kornberg. Recently, mutants lacking polymerase I (polA — ) have been identified. The investigations into the residual polymerase activity in polA — ceils have resulted in the isolation of two other DNA polymerases called polymerase II and polymerase III, in order of discovery. The three enzymes bear some similarities to the correspondingly numbered DNA polymerases from Escherichia coli . The three enzymes catalyze the covalent addition of nucleotides to a DNA primer under the direction of a DNA template using the four common deoxyribonucleoside 5'-triphosphates as substrate. The routine assay measures the incorporation of a radioactively labeled nucleoside triphosphate into a form that is insoluble in acid.
TL;DR: Data are presented indicating that inactivation at the non-permissive temperature (45’CO of thermolabile DNA polymerase III in a double polAI p0lC26~~ mutant of E. coli results in the failure to repair post-replication gaps after W-irradiation).
TL;DR: DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures and complementary RNA synthesized in vitro from C.fasciculATA kinetoplast DNA hybridizes with C. fascicULata nuclear DNA or Blastocrithidia culicis kinetOPlast DNA.
Abstract: SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH4)2SO4 and 0.13M (NH4)2SO4 respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn2+. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D.
Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil.
Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.
TL;DR: Findings may be interpreted to suggest that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or that many, if not all, DNA polymerases have different subsites for different templates.
TL;DR: Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis.
Abstract: Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis. Extracts of gently-lysed infected cells contain a DNA-dependent RNA polymerase activity which is specific for uracil-containing PBS2 DNA. The PBS2-induced RNA polymerase is insensitive to rifamycin derivatives which inhibit the host's RNA polymerase.
TL;DR: The template requirements of chick embryo DNA polymerase I devoid of nuclease activity, have been investigated and the high efficiency of hybrid structures rAn· dTn, dAn· rUn and rIn· dCn in which RNA acts as-initiator and DNA as template suggests an essential role of this enzyme in DNA replication.
Abstract: The template requirements of chick embryo DNA polymerase I devoid of nuclease activity, have been investigated. This enzyme catalyzes a repair reaction in vitro. Active templates were obtained by treatment of native DNA by DNAase I or exonuclease III. The DNA polymerase I activity requires divalent cations and is inhibited by high salt concentrations and sulfhydryl group blocking reagents.
Homopolydeoxynucleotides dAn, dTn, dCn, are efficient templates in the presence of hydrogen-bound initiator. The high efficiency of hybrid structures rAn· dTn, dAn· rUn and rIn· dCn in which RNA acts as-initiator and DNA as template suggests an essential role of this enzyme in DNA replication. The enzyme does not catalyze RNA-directed DNA synthesis.
DNA polymerase I of chick embryo and the avian myeloblastosis virus DNA polymerase were compared on the basis of their activities in the presence of various synthetic primers. Using synthetic DNA · RNA hybrid such as rAn· dTn, the cellular enzyme elongates the RNA strand by copying the DNA template whereas the viral enzyme mainly copies the RNA strand and less efficiently the DNA strand. With polymers of the (I + C) or (G + C) series, both enzymes are equally efficient which dCn as template; the viral enzyme activity can be distinguished from cellular enzymatic activity principally by its capacity to transcribe rCn and less efficiently rIn. The relationship between the responses of these enzymes in vitro to synthetic primers and the assumed physiological function of the cellular and viral DNA polymerases are discussed.