Showing papers on "Primase published in 1986"
TL;DR: Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species.
1,058 citations
TL;DR: It is demonstrated that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA.
585 citations
TL;DR: It is proposed that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagged strand.
476 citations
TL;DR: An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue, suggesting that the protein exists as a dimer of identical subunits.
460 citations
TL;DR: To simulate a reaction that occurs in T4-infected cells, an in vitro DNA synthesis system that requires seven highly purified proteins encoded by this bacteriophage is developed: the DNA polymerase "holoenzyme" (four proteins), gene 32 protein, dda DNA helicase, and uvsX protein - an enzyme that catalyzes homologueous DNA pairing and is functionally homologous to the recA protein.
251 citations
TL;DR: The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.
232 citations
Book•
01 May 1986
TL;DR: The authors present: General Introduction.
Abstract: The authors present: General Introduction. Multiplicity of DNA Polymerases in Animal Cells. DNA Polymerase-..cap alpha... DNA Polymerase-..beta... DNA Polymerase-..gamma.. and DNA Polymerase-delta. Roles of Animal Cell DNA Polymerases in Replication of Cellular DNA. Roles of Animal Cell DNA Polymerases in DNA Repair Synthesis. Fidelity of DNA Synthesis. Biology of Animal Cell DNA Polymerases. Future Perspectives. Index.
231 citations
TL;DR: The translocation of helicase II along single-stranded DNA is unidirectional and in the 3' to 5' direction with respect to the DNA strand on which the enzyme is bound.
206 citations
TL;DR: A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker.
Abstract: A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses.
177 citations
TL;DR: The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strander DNA as template.
Abstract: The plus strand of virion DNA of duck hepatitis B virus possessed, at its 5' terminus, a capped oligoribonucleotide 18 to 19 bases in length. This oligoribonucleotide had a unique 5' end, the heterogeneity in length reflecting two distinct junctions with plus-strand DNA that were 1 base apart. The sequence of the RNA differed from that predicted by the sequence of duck hepatitis B virus upstream of the 5' ends of plus-strand DNA but was identical to a downstream sequence corresponding to the 5' terminus of a major poly(A)+ viral RNA mapped by Buscher and co-workers (Cell 40:717-724, 1985). This RNA transcript is thought to serve as the template (i.e., the pregenome) for minus-strand synthesis via reverse transcription. The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strand DNA as template.
172 citations
TL;DR: It is proposed that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.
Abstract: The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.
TL;DR: The ColE1 primer transcript (RNA II) hybridizes to the template DNA near the replication origin and is cleaved by RNAase H to form the primer, which is inhibited by binding of another RNA, RNA I, to RNA II.
TL;DR: It is proposed that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calfDNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.
Abstract: Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.
TL;DR: Single-strand circular DNA from bacteriophage M13mp9 was chemically modified with osmium tetroxide to introduce specifically cis-thymine glycol lesions, a major type of DNA damage produced by ionizing radiation, which imply that the primary effect of the thymine gly col lesion is suppression of DNA synthesis and that the lesions is not a potent mutagen.
Abstract: Single-strand circular DNA from bacteriophage M13mp9 was chemically modified with osmium tetroxide to introduce specifically cis-thymine glycol lesions, a major type of DNA damage produced by ionizing radiation. An oligonucleotide primer was extended on damaged and undamaged templates using either the large fragment of E. coli pol I or T4 DNA polymerase. The reaction products were analysed by electrophoresis alongside a DNA sequence ladder. Synthesis on the damaged templates terminated at positions opposite thymine bases in the template. These results indicate that cis-thymine glycol lesions in single-strand DNA constitute blocks to synthesis by DNA polymerases in vitro. Surprisingly, replication halts after the correct nucleotide, dAMP, is inserted opposite the lesion. These results imply that the primary effect of the thymine glycol lesion is suppression of DNA synthesis and that the lesion is not a potent mutagen.
TL;DR: Like most eukaryotic SSB proteins, these proteins showed a 300-fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers.
Abstract: Affinity chromatography on double-stranded (ds) and single-stranded (ss) DNA-cellulose columns was employed to find analogs of the Escherichiu coli and T4 single-stranded DNA binding proteins (SSB proteins) in calf thymus. The interaction of several purified SSB proteins with the pure DNA-polymerase-cr - primase complex on DNA synthesis on activated DNA and on primase-initiated M13 DNA served as a criterion for a possible involvement of one of these proteins in the process of DNA replication. Two SSB proteins were purified to essential homogeneity. These most abundant proteins exhibited apparent relative molecular masses of 35 000 (SSB-35) and 37000 (SSB-37) for the protomers and 140000 and 80000 for the native enzymes. Both proteins resisted elution with 0.5 mg/ml dextran sulfate and were eluted from the ssDNA-cellulose with 0.2 M and 1 M NaCI, respectively. SSB-35 stimulated the DNA-polymerase-cl - primase complex from the same organism up to fivefold over a broad range of DNA covering. By contrast, SSB-37 inhibited the primase-initiated replication of M13 DNA. Like most eukaryotic SSB proteins, these proteins showed a 300-fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers. Furthermore, we provide evidence for a cooperative mode of binding for SSB-37. Although SSB35 and SSB-37 behave as typically eukaryotic SSB proteins in all assays employed, we tested these SSB proteins for dehydrogenase activities as well. SSB-35 was found to be identical with lactate dehydrogenase and SSB-37 was identical with a dimeric form of glyceraldehyde-3-phosphate dehydrogenase. These results imply that further studies are mandatory in order to prove the authenticity of eukaryotic SSB proteins. In the course of DNA replication, the double-stranded parental DNA has to be openend in order to provide a singlestranded template for the replicating enzyme, i.e. the DNA polymerase [ 11. These ssDNA intermediates will be covered in vivo by single-strand-specific DNA-binding proteins (SSB proteins). In prokaryotes and in several viruses the specific role of SSB proteins in the processes of DNA replication, DNA repair and DNA recombination has been widely clarified (for recent reviews see [2-41). By contrast, there is only a little information available on the function of SSB proteins in eukaryotes, despite the fact that SSB proteins from a variety of eukaryotic organisms have been isolated during the last decade [5 - 19,38 - 421 (and further references in [2 Since SSB proteins usually lack any enzymatic activity and since genetic studies on mammalian cells are not yet available,
TL;DR: It is established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons, which is stable in vivo and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerasealpha peptides in denaturing polyacrylamide gels.
TL;DR: Electrophoretic analysis of the electrophoretically homogeneous DNA polymerase alpha shows that it is composed of at least eight polypeptides in the molecular weight range of 180,000-15,000.
TL;DR: DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S has unusually high sedimentation coefficients, which are a result of association of the primase activity with RNA.
TL;DR: The ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II).
TL;DR: By raising the concentration of the 61 protein to a much higher level, a substantial amount of RNA-primed DNA synthesis is obtained in the absence of 41 protein, it is demonstrated that both pentaribonucleotide and tetraribon nucleotide primers can be used for efficient initiation of new DNA chains, whereas primers that are only 3 nucleotides long are inactive.
TL;DR: It is found that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA, relative to its activity on homopolymeric DNA.
Abstract: Purified calf thymus DNA polymerases delta I and II each have an associated 3' to 5' exonuclease but otherwise resemble DNA polymerase alpha in size, biochemical kinetic parameters, and the presence of DNA primase [Crute, J. J., Wahl, A. F., & Bambara, R. A. (1986) Biochemistry 25, 26-36]. Here we demonstrate a functional association of polymerase and exonuclease with each delta form. Furthermore, we show that the exonuclease can be dissociated from DNA polymerase delta I but does not appear to be removable from DNA polymerase delta II. Polymerases delta I, delta II, and alpha are equally sensitive to the inhibitor aphidicolin, suggesting a similarity in active site structure. In comparison with DNA polymerase alpha and delta II, DNA polymerase delta I has intermediate sensitivity to 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) or N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP). The activity of the DNA primase of the delta II enzyme is insensitive to BuAdATP whereas 1.0 microM of this inhibitor will decrease the activity of the DNA primase of the alpha and delta I enzymes approximately 50%. Two monoclonal antibodies that potently inhibit DNA polymerase alpha are only slightly inhibitory to DNA polymerase delta I and are ineffective at inhibiting DNA polymerase delta II. DNA polymerase delta II had been previously found to be nearly inactive on nuclease-treated calf thymus DNA, relative to its activity on homopolymeric DNA. We find that addition of purified calf histone proteins or spermidine can greatly enhance synthesis by this enzyme on activated calf DNA.
TL;DR: The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment and the complete nucleotide sequence of a 5 kb fragment revealed the presence of two other genes preceding rPOD in the order P23-dnaE-rpoD.
Abstract: The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.
TL;DR: A gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.
Abstract: Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established. The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate. Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction. Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.
TL;DR: These results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
Abstract: Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.
TL;DR: Yeast DNA primase-DNA polymerase activities catalyze de novo oligoribonucleotide primed DNA synthesis on single-stranded DNA templates and possible roles of DNA polymerase and DNAPrimase in RNA primer attenuation are discussed.
TL;DR: It is likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.
Abstract: We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution. Images
TL;DR: It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates.
TL;DR: It is suggested that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA Polymerase and the 55- and 68-k da peptides constituting the primase peptides.
TL;DR: Results suggest similar mechanisms of DNA synthesis for E. coli and T7 on the one hand and for phi 29 and adenovirus on the other.
TL;DR: Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell.
Abstract: The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.