Showing papers on "Primase published in 1978"

Analysis of unintegrated avian RNA tumor virus double-stranded DNA intermediates.

Abstract: Previous studies by Guntaka et al. have shown that the unintegrated DNA intermediates of avian RNA tumor virus replication can be readily isolated from cultures of the quail tumor line QT-6 at 1 day after infection. The intermediates include double-stranded linear and covalently closed circular DNA species. Using the analysis procedure of Southern together with previously obtained information regarding the sites of action of certain restriction endonucleases on avian sarcoma virus DNA, we have further characterized the viral DNA intermediates. Evidence is presented that, relative to the RNA genome, most of the linear species possess a direct terminal sequence redundancy equivalent to 0.5 X 10(6) +/- 0.3 X 10(6) daltons of double-stranded DNA. Some of the circular forms also possess a sequence redundancy of 0.21 X 10(6) +/- 0.03 X 10(6) daltons.

Mechanism of ultraviolet-induced mutagenesis: Extent and fidelity of in vitro DNA synthesis on irradiated templates

Abstract: The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded ϕX174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol+ and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated ϕX174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated ϕX174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3′→5′ “proofreading” exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase α, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis in bacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3′→5′ exonuclease activity associated with DNA polymerases.

Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication

Abstract: We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.

An RNA transcribed from DNA at the origin of phage fd single strand to replicative form conversion.

Abstract: 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.

Efficient correction of a mutation by use of chemically synthesized DNA.

Abstract: The mutated base in the am3 lysis-defective mutant of the bacteriophage phiX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 phiX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3(+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the phiX174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.

DNA synthesis : present and future

Abstract: I Initiation and Control of Replication.- Control of DNA Replication in Bacteria.- Map Position of the Replication Origin of the E. coli K12 Chromosome.- An Approach to the Isolation of the Replication Origin of Bacillus subtilis.- Stability of Origin-RNA and its Implications on the Structure of the Origin of Replication in E. coli.- The Genetics of E. coli DNA Replication.- Characterization of Chromosomal Replication in a dnaA Mutant.- Genetic and Physiological Properties of an Escherichia coli Strain Carrying the dnaA Mutation T46.- The Function of RNA Polymerase and dnaA in the Initiation of Chromosome Replication in Escherichia coli and Salmonella typhimurium.- Stabilized Initiation Activity of DNA Replication in an Sdre Mutant of Escherichia coli.- Replication of Bacteriophage Mu: Direction and Possible Location of the Origin.- Control of the Initiation of Lambda Replication, oop, lit and Repressor Establishment RNA Synthesis.- Control of Initiation of DNA Replication in Mammalian Cells.- Developmental Considerations of Eukaryotic DNA Replication and Replicon Size.- The Organization of DNA Replication in a Mammalian Cell Line.- An Unusual Structure Implicated in Initiation of DNA Synthesis.- Inhibition of DNA Synthesis by 1-?-D-arabino-furanosylcytosine: Differential Effect on Chain Initiation and Elongation in Human Lymphoblasts.- II The Biosynthesis of Chromosomal DNA.- Thymidine Metabolism in Bacteria (and "How, or How Not, to Label DNA").- Deoxyribonucleoside Triphosphates and DNA Polymerase in Bacteriophage PBS1-Infected Bacillus subtilis.- In Vivo Synthesis and Properties of Uracil-Containing DNA.- Excision Repair of Uracil in DNA and its Contribution to the Pool of Okazaki Fragments.- DNA Replication Intermediates in Escherichia coli.- Size Distribution of Short Chain DNA in Two Strains of Escherichia coli.- Evidence for the Absence of Triphosphate Termini from the 5' Ends of Newly Synthesized E. coli DNA Molecules.- On the Possible Direct Conversion of RNA to DNA.- III DNA Structure, DNA Binding and Unwinding Proteins.- Some Aspects of DNA Strand Separation.- Multiple Interactions of a DNA Binding Protein, Gene-32 Protein of Phage T4, During DNA Replication and Recombination.- Interaction of Protein HD from E. coli with Nucleic Acids.- A Putative DNA Gyrase Mutant of E. coli.- The Role of ATP in E. coli Chromosome Replication.- Nucleic Acid Binding Glycoproteins Which Solubilize Deoxyribonucleic Acid in Dilute Acid: Species Distribution and Possible Role in DNA Condensation.- ATP Dependent Deoxyribonuclease of Bacillussubtilis Subunit Structure and Substrate Specificity of DNA Dependent -ATPase Activity.- Pl Transduction Frequencies: A Clue to Chromosome Structure?.- Bacterial Nucleoid Structure After Inhibition of DNA Replication: The Role of RNA Synthesis.- Specific Properties of the End Groups of rDNA from Tetrahymena.- IV Polymerases and Other Replication Proteins.- Recombinational Mapping of the polA Locus of Escherichia coli K12: Genetic Fine Structure.- DNA Polymerase III of B. subtilis: Characterization of the Binding Site for Arylhydrazinopyrimidine Inhibitors.- DNA Proofreading by a Eukaryotic DNA Polymerase.- The Three DNA Polymerases of Animal Cells: Properties and Functions.- Structure and Catalytic Properties of Human DNA Polymerases ? and ?.- Properties and Interactions of DNA Polymerase ?, DNA Polymerase ? and a DNA Binding Protein of Regenerating Rat Liver.- DNA Polymerases of Sea Urchin Embryos.- Identity Between Nuclear and Mitochondrial DNA Polymerases ? from Chick Embryo.- Neurons as a Model System for In Vivo Studies of the Possible Function of DNA Polymerases in DNA Replication and Repair Synthesis During Development.- DNA Polymerase in Chromatin.- DNA Polymerase and the Onset of DNA Synthesis in Wheat Embryos.- Effect of SV40 Infection on DNA Polymerase Activity in Monkey Cells.- Characterization of Bacteriophage T7-Induced DNA Primase.- Suppression of E. coli dnaB Mutants by Prophage Plbac: A Biochemical Approach.- Two Replication Functions in Phage P1: ban, an Analog of dnaB, and bof, Involved in the Control of Replication.- V Viral and Plasmid Replication.- The Present Status of OX174 DNA Replication In Vivo.- Enzymatic Replication of DNA in E. coli Probed by Small Phages.- The dnaG Gene Product of Escherichia coli: Characterization of the Catalytic Activity.- G4 and ST-1 DNA Synthesis In Vitro.- Plasmid Replication.- Copy Number Control Mutants of the R Plasmid Rl in Escherichia coil.- Determination of Origin and Direction of Replication of Large Plasmids of E. coli by Two Electron Microscopic Techniques.- Replication of Colicin El Plasmid DNA In Vitro.- A Two-Stage Mechanism of Plasmid DNA Replication.- Adenoviruses - Model for DNA Replication in Mammalian Cells - Role of Viral Proteins in Viral DNA Replication.- Function of an Early DNA Binding Protein in the Replication of Adenovirus DNA.- Sequence Selectivity in the Integration of Adenovirus Type 2 DNA in Productively Infected Cells.- VI Repair Pathways.- DNA Repair in Human Cells: Enzymes and Mutants.- Enzymes Involved in the Repair of Damaged DNA.- In Vitro Characterization of the uvrA+, uvrB+, uvrC+-Coded ATP-Dependent UV-Endonuclease from Escherichia coli.- The recA+ Gene Product of Escherichia coli, an Inducible Protein.- UV Endonucleolytic Activity in Extracts from Mouse Cells.- On the Mechanism of Ultraviolet-Induced Mutagenesis.- An Adaptive Response of E. coli to Low Levels of Alkylating Agent.- Plasmid-Borne Error-Prone DNA Repair.- VII In Vitro Systems.- DNA Synthesis in Permeable Cell Systems from Saccharomyces cerevisiae.- DNA Replication in Simple Eukaryons: Identification of Replisomal Complexes in Cell Extracts of Chlamydomonas reinhartii.- Studies with Enzyme Complexes that Synthesize Adenovirus 2 DNA In Vitro.- Studies on Replicating Simian Virus 40 Chromosomes: Association with Thymidine Kinase.- Synthesis of DNA and Poly(ADP Ribose) in Permeabilized Eukaryotic Cells.- Effect of Xenopus laevis Oocyte and Egg Extracts on SV40 DNA.

beta-Lapachone, an inhibitor of oncornavirus reverse transcriptase and eukaryotic DNA polymerase-alpha. Inhibitory effect, thiol dependence and specificity.

Abstract: beta-Lapachone is a naturally occuring compound that can be isolated from a number of tropical trees. It is shown to be a potent inhibitor of reverse transcriptase activity from both avian myeloblastosis virus and Rauscher murine leukaemia virus. In addition, it affects eukaryotic DNA-dependent DNA polymerase-alpha activity: 50% inhibition is reached in 60-min incubation time by about 8 micron beta-lapachone. Enzyme activity is inhibited irrespective of the purity of the enzyme used or of the amount or type of template/primer or substrate present. The inhibitory effect of the drug is only observed in the presence of dithiothreitol. The primary site of action of beta-lapachone appears to be the enzyme protein, as is also borne out by the specificity of its action. Eukaryotic DNA-dependent DNA polymerase-beta, prokaryotic DNA-dependent DNA polymerase I, several other nucleic acid polymerases and some completely unrelated enzymes are not affected. Reverse transcriptase and DNA-dependent DNA polymerase-alpha may be in someway related in possessing similarly exposed '--SH structures' in their active sites. beta-lapachone thus affords a novel means of studying such interrelationships and of further characterizing enzymes.

Photochemical cross-linking studies on the interaction of Escherichia coli RNA polymerase with T7 DNA

Abstract: We have identified the subunits of Escherichia coli RNA polymerase which are in close contact with the T7 phage DNA template using photochemical cross-linking. In nonspecific T7 DNA-enzyme complexes which occur in all regions of the DNA, subunits sigma, beta, and beta' were cross-linked to the DNA. In contrast, in specific binary complexes which presumably occur at promoter sites, and in the initiation complex (holoenzyme + T7 DNA + initiator dinucleotides + three nucleoside triphosphates), only sigma and beta were cross-linked to DNA, while cross-linking of beta' could not be demonstrated. These results (1) do not support the idea that alpha subunits are involved in the enzyme-template interaction, (2) raise the possibility that sigma subunit participates directly in promoter recognition even though isolated sigma does not bind to DNA, and (3) indicate different modes of interaction between RNA polymerase and DNA in nonspecific and specific complexes. These findings are relevant to the mechanism by which RNA polymerase carries out selective transcription.

A study of unwinding of DNA and shielding of the DNA grooves by RNA polymerase by using methylation with dimethylsulphate

Abstract: The dimethylsulphate method has been used to study the complexes of RNA polymerase (Escherichia coli) with DNA of T7 phage, poly[d(A--T)] and fragments of calf thymus DNA protected against DNase digestion by RNA polymerase. The binding of RNA polymerase to DNA significantly increases the formation of 1-methyl-adenine produced by methylation of the single-stranded DNA region, diminishes by about 10% the formation of 3-methyl-adenine by methylation within the minor groove and does not affect the formation of 7-methyl-guanine by methylation within the major DNA groove. The presence of nascent RNA decreases the formation of 1-methyl-adenine in DNA of the complex by about 30%. The initiation of RNA synthesis or RNA synthesis itself does not influence the methylation of the major groove but shielding of the minor groove increases by about twice as much. These results suggest that RNA polymerase, upon binding, breaks Watson-Crick base-pairing in a DNA region of about 15-base-pairs long, that nascent RNA forms a duplex with DNA of about 10-base-pairs long; and that the enzyme weakly interacts with DNA along its grooves and preferentially makes contacts with the minor groove.

HeLa DNA polymerase ∝ activity in vitro: specific stimulation by a non-enzymic protein factor

Abstract: A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.

Recently replicated simian virus 40 DNA is a preferential template for transcription and replication.

Abstract: The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.

Yeast DNA polymerases: antigenic relationship, use of RNA primer and associated exonuclease activity.

Abstract: Highly purified preparations of DNA polymerases A and B from yeast were compared with respect to antigenic relationship, ability to use ribonucleotide primers and associated nuclease activity The following results were obtained 1 Antiserum directed against DNA polymerase A inhibits this enzyme but does not interfere with the activity of DNA polymerase B or of mitochondrial DNA polymerase, nor does it precipitate the latter two enzymes 2 DNA polymerase A is capable of using oligo(ribouridylic acid) as a primer for the polymerization of dTMP This reaction is not catalysed by polymerase B to any significant extent 3 Whereas DNA polymerase A is devoid of nuclease activity, DNA polymerase B catalyses an exonucleolytic release of mononucleotide units from the 3′ end of polynucleotides The results of several experiments suggest that this nuclease activity is associated with the DNA polymerase B molecule

Primase initiates Okazaki pieces during polyoma DNA synthesis.

Abstract: SHORT fragments (Okazaki pieces) are formed as intermediates during elongation of nascent DNA strands1. In isolated nuclei from polyoma-virus infected 3T6 cells Okazaki pieces are initiated by a short RNA primer (initiator RNA, iRNA) with the approximate size of a decanucleotide2. The nature of the polymerase responsible for primer synthesis is not known, but high concentrations of α-amanitin, a drug which inhibits both RNA polymerase II (ref. 3) and III (ref. 4), have no effect on primer formation (unpublished results). We now present evidence that iRNA is not synthesised by any of the known RNA polymerases, but that, instead, a mammalian enzyme is involved which in its action corresponds to the dnaG gene product of E. coli. The bacterial enzyme was named primase5. It participates in the initiation of DNA strands of some single-stranded bacteriophages and has been suggested to be involved in the synthesis of bacterial Okazaki pieces6,7. One of the distinguishing features of the E. coli enzyme is its ability to use both ribonucleotides and deoxyribonucleotides for primer synthesis5,8.

Initiation of HeLa cell DNA synthesis in a subnuclear system

Abstract: Mammalian cells are known to synthesize DNA in discrete stages, the first of which seems to be the formation of DNA pieces 150--200 nucleotides in length that have a s20 value of about 4 S. We have reconstructed a system derived from HeLa cell nuclei that carries out RNA-primed initiation of the synthesis of small (4S) DNA fragments. This synthesis is resistant to high concentrations of alpha-amanitin and sensitive to antibody directed against RNA polymerase I, suggesting that this enzyme may be involved in the initiation step. The formation of small DNA fragments in this system also requires DNA polymerase alpha, heat-labile nuclear factor(s), and at least one other nuclear protein.

Adenovirus DNA synthesis in vitro in an isolated complex.

Abstract: DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.

Initiation of enzymatic DNA synthesis by yeast RNA polymerase I.

Abstract: In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.

Subunit topography of RNA polymerase (E. coli) in the complex with DNA

Abstract: E. Coli RNA polymerase binding to different DNAs (from E. Coli, 5-bromodeoxyuridine (BrdUrd) substituted DNA and poly [d(BrU-A)] was induced with ultraviolet (U.V.) light to form protein-DNA crosslinked complexes. Two independent methods of analysis, polyacrylamide gel electrophoresis in SDS and chloroform extraction indicated the formation of a stable complex between the enzyme and DNA. The complexes were formed under different ionic strength conditions, at low enzyme to DNA ratios in order to approach the conditions of specific binding. In contrast there was no crosslinking of the complex in 1 M KCl solution which dissociates the enzyme from DNA. The efficiency of formation of strongly bound complex was found to be much higher with holoenzyme than with core enzyme. The following results were obtained : 1) The large subunits beta and beta' were found to be bound to DNA. 2) Relatively small amount of sigma subunit were bound to DNA while alpha subunits were essentially not attached to DNA. The high binding affinity of beta and beta' subunits was also observed in the studies of isolated subunits. These results lead to a model of enzyme-DNA complex in which the large beta and beta' subunits provide the contacts between the RNA polymerase and the DNA.

In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome.

Abstract: Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.