Showing papers on "Primase published in 1988"

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase.

Abstract: We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.

Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.

Abstract: Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.

Structures of herpes simplex virus type 1 genes required for replication of virus DNA.

Abstract: Recently, a method has been developed to identify regions in the genome of herpes simplex virus type 1 (HSV-1) which contain genes required for DNA synthesis from an HSV-1 origin of DNA replication, and seven genomic loci have been identified as representing the necessary and sufficient gene set for such replication (C. A. Wu, N. J. Nelson, D. J. McGeoch, and M. D. Challberg, J. Virol. 62:435-443, 1988). Two of the loci represent the well-known genes for DNA polymerase and major DNA-binding protein, but the remainder had little or no previous characterization. In this report we present the DNA sequences of the five newly identified genes and their deduced transcript organizations and encoded amino acid sequences. These genes were designated UL5, UL8, UL9, UL42, and UL52 and were predicted to encode proteins with molecular weights of, respectively, 99,000, 80,000, 94,000, 51,000, and 114,000. All of these genes had clear counterparts in the genome of the related alphaherpesvirus varicella-zoster virus, but only UL5 and UL52 were detectably conserved in the distantly related gammaherpesvirus Epstein-Barr virus, as judged by amino acid sequence similarity. The sequence of the UL5 protein, and of its counterparts in the other viruses, contained a region closely resembling known ATP-binding sites; this could be indicative, for instance, of a helicase or primase activity.

Generation of telomere-length heterogeneity in Saccharomyces cerevisiae.

Abstract: Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats. We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication. For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone. This type of variation was independent of the major yeast recombination pathway. The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication. Lengths of individual telomeres within a single clone varied independently of each other. Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately. These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication.

Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase.

Abstract: Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.

In situ transcription in cells and tissues

Abstract: In situ transcription is provided by hybridizing cells or tissue with a primer, and extending the primer bound to any template mRNA or DNA with transverse transcriptase or DNA polymerase in the presence of labeled nucleotides, which allows for detection of cells containing the template. The resulting cDNA may be eluted and used in a polymerase chain reaction for isolation of the DNA, and/or the cDNA electrophoresed, which may provide for information concerning the sequence, or the like.

A DNA helicase induced by herpes simplex virus type 1

Abstract: We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.

A 7-kDa region of the bacteriophage T7 gene 4 protein is required for primase but not for helicase activity.

Abstract: Bacteriophage T7 gene 4 protein, purified from phage-infected cells, consists of a mixture of 56- and 63-kDa species that provides helicase and primase activities required for T7 DNA replication. The 56-kDa species has been purified independently of the colinear 63-kDa species. Like a mixture of the two proteins, the 56-kDa protein binds single-stranded DNA in the presence of dTTP, catalyzes DNA-dependent hydrolysis of dTTP, and has helicase activity. In contrast to the mixture, the 56-kDa protein cannot catalyze template-dependent RNA primer synthesis. In the absence of a DNA template, both the 56-kDa protein and the mixture of the two species synthesize low levels of diribonucleotide. A putative "zinc finger" present near the amino terminus of the 63-kDa protein but absent from the 56-kDa protein may play a major role in the recognition of primase sites in the template.

Differential inhibition of various deoxyribonucleic and ribonucleic acid polymerases by suramin.

Abstract: The inhibitory effects of hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4,6, 8-trisulfonate)carbamide (trivial name: suramin) on the activities of various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases from mammalian cells, bacteria and retrovirus were examined and compared with each other. Among the various DNA and RNA polymerases tested, the activities of DNA primase, DNA polymerase alpha, reverse transcriptase and Escherichia coli RNA polymerase were strongly inhibited by suramin, while the activities of other enzymes including DNA polymerases beta and gamma, terminal deoxynucleotidyl-transferase and DNA polymerase I were relatively resistant to inhibition by this drug. The inhibition by suramin of DNA polymerase alpha from KB cells and Rauscher murine leukemia virus (RLV) reverse transcriptase was due to competition with the respective template primer (activated DNA for alpha polymerase and (rA)n.(dT)12-18 for reverse transcriptase) for the template.primer-binding site of the enzyme, while the inhibition of DNA primase and E.coli RNA polymerase was due to competition with the ribonucleoside triphosphate substrate. The inhibition constants (Ki) of suramin were determined to be 2.6 microM, 0.35 microM, 0.54 microM and 0.70 microM for DNA primase, DNA polymerase alpha, RLV reverse transcriptase and E. coli RNA polymerase respectively. The observed inhibitions of these polynucleotide-synthesizing enzymes by suramin seem to explain, at least in part, an as yet unknown mechanism of trypanocidal action of this drug.

Mammalian cyclin/PCNA (DNA polymerase δ auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III

Abstract: Human cyclin/PCNA (proliferating cell nuclear antigen) is structurally, functionally, and immunologically homologous to the calf thymus auxiliary protein for DNA polymerase delta. This auxiliary protein has been investigated as a stimulatory factor for the nuclear DNA polymerases from S. cerevisiae. Calf cyclin/PCNA enhances by more than ten-fold the ability of DNA polymerase III to replicate templates with high template/primer ratios, e.g. poly(dA).oligo(dT) (40:1). The degree of stimulation increases with the template/primer ratio. At a high template/primer ratio, i.e. low primer density, cyclin/PCNA greatly increases processive DNA synthesis by DNA polymerase III. At low template/primer ratios (e.g. poly(dA).oligo(dT) (2.5:1), where addition of cyclin/PCNA only minimally increases the processivity of DNA polymerase III, a several-fold stimulation of total DNA synthesis is still observed. This indicates that cyclin/PCNA may also increase productive binding of DNA polymerase III to the template-primer and stabilize the template-primer-polymerase complex. The activity of yeast DNA polymerases I and II is not affected by addition of cyclin/PCNA. These results strengthen the hypothesis that yeast DNA polymerase III is functionally analogous to the mammalian DNA polymerase delta.

Sites of termination of in vitro DNA synthesis on cis-diamminedichloroplatinum(II) treated single-stranded DNA: a comparison between E.coli DNA polymerase I and eucaryotic DNA polymerases α

Abstract: We have compared the capacity of the large fragment of E. coli DNA polymerase I and highly purified DNA polymerases alpha from either Drosophila melanogaster embryos or calf thymus to replicate single-stranded M13 mp10 DNA treated with the antitumoral drug cis-diamminedichloroplatinum(II) (cis-DDP). We report that: a) although prokaryotic and eukaryotic enzymes have different structural complexity and dissimilar in vivo functions, their synthesis was blocked in vitro at similar sites on cis-DDP treated DNA; b) this inhibition occurred not only at d(G)n sequences, as previously reported for E. coli DNA polymerase I, (Pinto & Lippard (1985) Proc. Natl. Acad. Sci. USA, 82, 4616-4619) but also at other sequences which may represent putative cis-DDP-DNA adducts.

Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid.

Abstract: The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide-synthesizing enzymes were examined. Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases α, β, γ, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase. ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase α. Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template · primer for the same binding site of the enzyme. On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template · primers or nucleoside 5′ -triphosphate substrates. The inhibition constants for ATA were, in general, smaller than those for Evans blue.

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains.

Abstract: It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.

Calf thymus DNA polymerase δ: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase α, a DNA dependent ATPase and proliferating cell nuclear antigen

Abstract: We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.

Further characterization of DNA helicase activity of mouse DNA-dependent adenosinetriphosphatase B (DNA helicase B).

Abstract: The DNA helicase activity of DNA-dependent ATPase B purified from mouse FM3A cells [Seki, M., Enomoto, T., Hanaoka, F., & Yamada, M. (1987) Biochemistry 26, 2924-2928] has been further characterized. The helicase activity was assayed with partially duplex DNA substrates in which oligonucleotides to be released by the enzyme were radiolabeled. Oligonucleotides with or without phosphate at the 5' termini or with a deoxy- or dideoxyribose at the 3'-terminal nucleotides were displaced by this enzyme with essentially the same efficiency and with the same ATP (and dATP) and Mg2+ requirements. Thus, there was no strict structure requirement for both ends of duplex regions of substrates to be unwound by the enzyme. Shorter strands were released more readily than longer strands up to the length of 140 bases. The attachment of the enzyme to a single-stranded DNA region was a prerequisite for the neighboring duplex to be unwound; the enzyme-catalyzed unwinding was inhibited competitively by the coaddition of single-stranded DNAs which act as cofactors of the ATPase activity. Their activities as the inhibitor of helicase were well correlated with those as the cofactor of ATPase. The helicase B was found to migrate along single-stranded DNA in the 5' to 3' direction by the use of single strands with short duplex regions at both 3' and 5' ends as substrate. A possible role of this enzyme in DNA replication in mammalian cells is discussed.

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperaturesensitive mutant altered in DNA primase-polymerase complex stability

Abstract: The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.