Showing papers on "Primase published in 1989"
TL;DR: In this article, it was shown that the phi 29 DNA polymerase is the only enzyme required for efficient DNA replication with the initiation primer as the only additional protein requirement, and that conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme.
723 citations
TL;DR: The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.
Abstract: Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis. Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate. Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage. A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis. The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.
298 citations
TL;DR: The three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.
Abstract: In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.
232 citations
TL;DR: It is suggested that co‐ordinated synthesis of these strands requires dynamic protein‐protein interactions between these replication factors and the two DNA polymerases.
Abstract: DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase alpha activity 4- to 6-fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co-operatively by PCNA, RF-A and RF-C approximately 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.
219 citations
TL;DR: The primary sequence of human DNA polymerase α deduced from the full‐length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage ɸ19, yeastDNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus.
Abstract: The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.
187 citations
TL;DR: The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enablednaB to perform its ATPase, helicase, and priming functions.
144 citations
TL;DR: Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reass association of the DNA with the exonuclease site of a second molecule of Klenow fragments.
128 citations
TL;DR: Large T antigen, the regulatory protein encoded by simian virus 40, has DNA helicase activity and unwinds double-stranded DNA at the expense of ATP, and it appears that the bound nucleotide determines whether T antigen acts as an RNA helicase or as aDNA helicase.
122 citations
TL;DR: The results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.
Abstract: A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.
99 citations
TL;DR: It is shown here that the presence of 41 protein on the lagging strand of the fork enables the polymerase holoenzyme to catalyze leading strand DNA synthesis at a maximum rate and with high processivity.
95 citations
TL;DR: It is found that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail.
TL;DR: An in vitro system, composed of highly purified bacteriophage lambda and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the lambda replication origin (ori lambda), concludes that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted lambda replication system.
TL;DR: It is found that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene.
TL;DR: Results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo.
Abstract: We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.
Patent•
[...]
24 Mar 1989
TL;DR: An improved method for determining the nucleotide base sequence of a DNA molecule was proposed in this paper, which includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecules, incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific base.
Abstract: An improved method for determining the nucleotide base sequence of a DNA molecule. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA-polymerase which is a φ29-type DNA polymerase.
TL;DR: This chapter discusses the DNA polymerases α and δ from mammalian cells and their accessory factors, and on the analogous DNA polymerase from Drosophila melanogaster and Saccharomyces cerevisiae, and a comparison of the polymerases from these organisms is problematic for several reasons that are described in the chapter.
Abstract: Publisher Summary This chapter discusses the DNA polymerases α and δ from mammalian cells and their accessory factors, and on the analogous DNA polymerases from Drosophila melanogaster and Saccharomyces cerevisiae . DNA-polymerizing activity in eukaryotic tissues was discovered not long after Kornberg's discovery of DNA polymerase I from Escherichia coli. The gene for human DNA polymerase α has been isolated and characterized, and new high-fidelity forms of the enzyme have been purified, using rapid isolation techniques. DNA polymerase δ has gained considerable attention because of its interaction with a cell-cycle-regulated protein. This protein, proliferating cell nuclear antigen (PCNA), or cyclin, is also required for in vitro DNA replication of simian-virus-40 (SV40) DNA replication. The major differentiating characteristics of the four mammalian DNA polymerases are summarized in the chapter through a table. DNA polymerases have been isolated from a variety of unicellular eukaryotes. A comparison of the polymerases from these organisms with the prototypical mammalian DNA polymerases is problematic for several reasons that are described in the chapter.
TL;DR: Results suggest that the polymerase and exonuclease activities are physically associated, and it remains to be determined if they reside in the same subunit.
Abstract: Porcine liver DNA polymerase gamma contains exonuclease activity capable of digesting DNA in the 3'----5' direction, releasing deoxyribonucleoside 5'-monophosphates. The exonuclease activity excises 3'-terminal bases from both matched and mismatched primer termini, with a preference for mismatched bases. Under polymerization conditions, mismatch excision by the exonuclease occurs prior to polymerization by polymerase gamma, and this excision can be inhibited by adding to the reaction a high concentration of dNTP substrates and/or nucleoside 5'-monophosphates. In an M13mp2-based reversion assay for detecting single-base substitution errors, porcine liver polymerase gamma is highly accurate; the estimated base substitution error rate is less than one error for each 500,000 bases polymerized. Lower fidelity is observed using reaction conditions that inhibit the exonuclease activity, strongly suggesting that the exonuclease proofreads errors during polymerization. However, in a forward mutation assay capable of detecting all 12 mispairs at a variety of template positions, certain base substitution errors are readily detected even using unperturbed polymerization conditions. Thus, for some errors, polymerase gamma is not highly accurate, suggesting that proofreading is not equally active against all mispairs. To examine if the polymerase and exonuclease activities are physically as well as functionally associated, both activities were monitored during purification by four procedures, each based on a different separation principle. The two activities copurify during chromatography using phosphocellulose, heparin-agarose, or double-strand DNA-cellulose, and during velocity sedimentation in a glycerol gradient containing 0.5 M KCl. These results suggest that the polymerase and exonuclease activities are physically associated. It remains to be determined if they reside in the same subunit.
TL;DR: The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.
Abstract: DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.
TL;DR: The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells and catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-k da protein.
TL;DR: A clustering of sites is observed, suggesting that the polymerase holoenzyme may become destabilized when moving along selected regions of the DNA and then pause at one or more of several closely spaced positions.
TL;DR: Evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme.
TL;DR: It is proposed that segments A-D of the M2 and phi 29 DNA polymerases constitute a structure which forms the cleft for holding template DNA and that segment D is a region for interacting with dNTP.
TL;DR: Azidothymidine triphosphate acted as an efficient inhibitor of cellularDNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerases beta, were not inhibited.
Abstract: The reverse transcriptase from human immunodeficiency virus type 1 was purified from the virus to near homogeneity. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA-synthesizing activity. Activated DNA as a heteropolymeric substrate was used as efficiently as was the homopolymeric substrate poly(rA)-oligo(dT). The Michaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Azidothymidine triphosphate, a well-known inhibitor of the enzyme, inhibited the enzyme competitively with respect to dTTP and noncompetitively with respect to the other nucleotides. Azidothymidine triphosphate acted as an efficient inhibitor of cellular DNA polymerase gamma, whereas other enzymes of eucaryotic DNA metabolism, namely, DNA polymerase alpha-primase and DNA polymerase beta, were not inhibited. This finding may explain why some acquired immunodeficiency syndrome patients suffer side effects during azidothymidine therapy.
TL;DR: This report shows that in the absence of their cognate promoter, two bacteriophage RNA polymerases are capable of performing unusual transcription reactions: (i) the DNA template serves also as a primer for RNA synthesis and this leads to hybrid DNA/RNA molecules, and (ii) if theDNA template forms a hairpin structure, the linear DNA can be transcribed via the 'rolling circle' mechanism.
Abstract: Efficient transcription reactions of DNA-dependent RNA polymerases require the presence of a specific promoter sequence. This report shows that in the absence of their cognate promoter, two bacteriophage RNA polymerases are capable of performing unusual transcription reactions: (i) the DNA template serves also as a primer for RNA synthesis and this leads to hybrid DNA/RNA molecules, (ii) if the DNA template forms a hairpin structure, the linear DNA can be transcribed via the 'rolling circle' mechanism.
TL;DR: A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae and a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex is proposed.
TL;DR: The results suggest that the 3' end of the RNA primer is determined prior to its transfer to the primer-binding site and that the determination of the 3', not the 2' end, of the primer does not depend on a specific sequence motif at the cleavage site.
Abstract: The replication of the hepadnavirus DNA genome is initiated by reverse transcription of pregenome RNA into minus-strand DNA followed by plus-strand DNA synthesis. The priming of plus-strand DNA requires the transfer of an RNA primer from pregenome RNA to the primer-binding site on minus-strand DNA. Annealing of the primer to the primer-binding site is facilitated by short direct repeats, DR1 and DR2. To investigate the mechanism of plus-strand primer formation, we have introduced specific mutations into DR1 and DR2 and measured the effect of these mutants on initiation of plus-strand DNA synthesis. To facilitate such an analysis, we have constructed a vector for the efficient expression of woodchuck hepatitis virus in cultured cells. Our results suggest that the 3' end of the RNA primer is determined prior to its transfer to the primer-binding site and that the determination of the 3' end of the primer does not depend on a specific sequence motif at the cleavage site. In addition, we have identified an alternative initiation site for plus-strand DNA synthesis at a purine-rich sequence between DR1 and DR2. Initiation at this site occurs by a mechanism that is independent of the direct repeats and does not require the transfer of an RNA primer to the primer-binding site.
TL;DR: The purified DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius has neither exonuclease nor primase activities and is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin.
Abstract: We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2′,3′-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80°C and is active between 55°C and 85°C in the presence of activated calf-thymus DNA.
TL;DR: It is proposed that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T 4 replication complex.
TL;DR: A mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA Polymerase alpha.
Abstract: Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.
TL;DR: The 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe but not in RNA from hamster or human cells, indicating a conserved 5' portion and divergent 3' region of the expressed gene.