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At least 217 records · Page 12Linked to original sources
PubMed1984

Plasmid-phage recombination in T7 infected Escherichia coli.

Recombination between genetically marked T7 bacteriophage and plasmids containing inserts of T7 DNA has been studied in order to gain some insight into the phage recombination process. The results suggest that plasmid-phage recombination requires the products of T7 genes 3 (endonuclease), 4 (DNA primase), 5 (DNA polymerase), and 6 (exonuclease), as has been demonstrated previously for phage-phage recombination. Plasmid replication does not compensate for a complete block in phage polymerase synthesis, suggesting a direct role for this enzyme in recombination, rather than an indirect role, by means of producing replicative structures that are recombinogenic. In most respects, plasmid-phage recombination appears to be similar to phage-phage recombination. The participation of two autonomous, structurally dissimilar, homologues, however, might render certain aspects of the recombination process more amenable to analysis. As examples, the characterization of an apparent marker effect and the demonstration of genetic heterozygotes among the products of plasmid-phage recombination are presented.

DNA Primase↗
PubMed1988

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

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.

Chromosome Mapping↗
PubMed1985

Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.

Animals↗
PubMed2002

Maize replicative alpha-type DNA polymerase: separation of polymerase and primase activities and recognition of primase subunits.

DNA polymerase and DNA primase activities in the maize alpha-type DNA polymerase 2 were dissociated and DNA polymerase-free DNA primase was studied. DNA primase synthesized primers that were 8-34 nucleotides long, with more intense bands at 15-17 nucleotides in length. DNA polymerase 1 (a putative delta-type enzyme) or DNA polymerase 2 were assayed after template-priming with purified DNA primase and showed a differential use of templates: whereas DNA polymerase 2 used a polydT template more efficiently than a natural template, DNA polymerase 1 used both of them poorly. The molecular size of DNA primase was estimated to be 68 kDa by gel filtration, western blotting and by a DNA primase 'trapping' assay.

Journal Article↗
PubMed1989

A Ca2+-dependent DNA primase-like activity from HeLa cells.

Novel DNA primase-like activity was partially purified from human Hela cells, and the activity was clearly separated from DNA polymerase alpha by phosphocellulose column chromatography. The enzyme did not show significant activity in the absence of Ca2+, and was dramatically activated by the addition of Ca2+; so it was designated as C-primase. The C-primase showed a molecular weight of 20,000 estimated by gel filtration and a sedimentation coefficient of 3.5 S by glycerol gradient centrifugation. These results, together with the other properties of the C-primase, suggest that this primase like-enzyme is distinct from the authentic eukaryotic primase in the DNA polymerase alpha/DNA primase complex.

Calcium↗
PubMed1993

An error-correcting proofreading exonuclease-polymerase that copurifies with DNA-polymerase-alpha-primase.

A DNA polymerase with a 3'-to 5'-exonuclease that copurified with polymerase-primase from calf thymus was purified and extensively characterized. Its exonuclease degraded single-stranded DNA from 3' to 5' in a strictly distributive manner. On synthetic template-primer junctions, 3'-terminal mispairs were excised with a 10- to 20-fold preference over correctly paired nucleotides. In comparison to the 3'- to 5'-exonuclease the DNA polymerase activity was rather low. The ratio of nucleotides incorporated to nucleotides excised was in the order of 1 to 3 nucleotide insertions per excision, suggesting that net forward DNA synthesis is not the primary role of this DNA polymerase. DNA synthesis was performed with a low processivity in the presence and absence of PCNA. Both the polymerase and exonuclease activities were inhibited to a comparable extent by AMP. Thus, the exonuclease-polymerase might represent a novel DNA polymerase that we tentatively designate as DNA polymerase zeta. Possible benefits of DNA polymerase zeta in the process of error correction and the apparent dichotomy of an built-in proofreading activity for the processive DNA polymerases gamma, delta, and epsilon and an obviously external proofreading function for the less processive animal cell DNA polymerases alpha and beta are discussed.

Animals↗
PubMed2001

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.

Amino Acid Motifs↗
PubMed1985

Inhibition of the activities of DNA primase-polymerase alpha complex from KB cells by hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4 ,6,8-trisulfonate)carbamide.

A trypanocidal drug suramin [hexasodium sym-bis(m-amino-benzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4, 6, 8-trisulfonate)carbamide] was found to be a potent inhibitor of the activities of DNA primase and polymerase alpha from human KB cells. The mechanism of the inhibition by suramin was, however, quite different by these two polymerases. In the case of DNA primase, suramin inhibited competitively the incorporation of a nucleotide substrate, GTP, on the template polydeoxycytidylate, while the polymerase alpha was inhibited competitively by the drug with respect to the template primer (activated DNA). The observed inhibitory effect of suramin on nucleic acid synthesis seems to explain yet unknown mechanism of trypanocidal action of the drug.

Binding, Competitive↗
PubMed1997

The DNA polymerase alpha-primase complex couples DNA replication, cell-cycle progression and DNA-damage response.

The highly conserved DNA polymerase alpha-primase complex (pol-prism) is the only eukaryotic DNA polymerase that can initiate DNA synthesis de novo. It is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. The dual role of pol-prim makes it a likely target for mechanisms that control cell-cycle S-phase entry and progression.

Animals↗
PubMed1998

Simultaneous formation of functional leading and lagging strand holoenzyme complexes on a small, defined DNA substrate.

The biochemical characterization of leading and lagging strand DNA synthesis by bacteriophage T4 replication proteins has been addressed utilizing a small, defined primer/template. The ATP hydrolysis activity of 44/62, the clamp loading complex responsible for holoenzyme assembly, was monitored during assembly of both the leading and lagging strand holoenzyme complex. The ATPase activity of 44/62 diminishes once a functional holoenzyme is assembled on both the leading and lagging strand. The assembly of the lagging strand holoenzyme is facilitated by several factors including biotinylated streptavidin blocks at the end of the fork strands, preassembly of the leading strand holoenzyme, and by the presence of the DNA primase with ribonucleoside triphosphates. The resultant minimal replicative complex consists of two holoenzymes and a primase nested on a model replication fork derived from a 62-mer template/34-mer primer/36-mer lagging strand in an apparent 2:2:1:1 ratio of 45 protein:polymerase:primase:forked DNA. The 44/62 protein complex does not remain associated with the complex. The primase alone slowly synthesizes pentaribonucleotides on the forked DNA when the lagging strand contains a nonannealed TTG initiation site with the rate of synthesis greatly stimulated by the addition of the 41 helicase. The addition of deoxy-NTPs to this complex results in leading strand synthesis, but extension of the synthesized RNA primer does not occur. DNA synthesis in both the leading and lagging strand directions is achieved, however, when a 6-mer DNA primer is annealed to the primase recognition site of the forked DNA substrate. A model is presented that describes how leading and lagging strand DNA synthesis might be coordinated as well as the associated molecular interactions of the replicative proteins.

Adenosine Triphosphatases↗
PubMed2001

Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro.

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

Amino Acid Sequence↗
PubMed1996

The UL8 subunit of the herpes simplex virus type-1 DNA helicase-primase optimizes utilization of DNA templates covered by the homologous single-strand DNA-binding protein ICP8.

The herpes simplex virus type-1 DNA helicase-primase is a heterotrimer encoded by the UL5, UL8, and UL52 genes. The core enzyme, specified by the UL5 and UL52 genes, retains DNA helicase, DNA-dependent nucleoside triphosphatase, and primase activities. The UL8 subunit has previously been implicated in increasing primer stability and in stimulating primer synthesis by the core enzyme. To further characterize the function of the UL8 subunit, we have examined its effect on the activities of the UL5/52 core enzyme using DNA templates covered by the herpes simplex virus type-1 single-strand DNA-binding protein ICP8. We found that while ICP8 stimulated the DNA helicase activity of the UL5/52 proteins up to 3-fold, maximum stimulation by ICP8 required the presence of UL8 protein. Moreover, UL8 protein was required to reverse the inhibitory effect of ICP8 on the DNA-dependent ATPase and primase activities of the UL5/52 proteins. These observations were specific for ICP8 since the heterologous Escherichia coli single-strand DNA-binding protein could not substitute for ICP8. These data suggest that UL8 protein mediates an interaction between the UL5/52 core enzyme and ICP8 that optimizes the utilization of ICP8-covered DNA templates during DNA replication.

Animals↗
PubMed1996

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination.

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors. DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase alpha. Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a delta-like activity.

Animals↗
PubMed1988

Replication of single-stranded porcine circovirus DNA by DNA polymerases alpha and delta.

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64-66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39-57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789-4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.

Animals↗
PubMed2001

Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.

Activities of the primase (Pr)-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring reaction systems represented by special NP complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (J. Riman, A. Sulová and K. Horská, Acta virol. 39, 149-159 (1995); J. Ríman and A. Sulová, Acta virol. 41, 181-192 (1997)) were studied in the absence and presence of carbonyldiphosphonate (COMDP), mimosine (MIMO), to it related ciclopirox olamine (CPX) and butylphenyl deoxyguanosine-5'-triphosphate (BuPdGTP). Reaction products radioactively labeled for RNA and DNA and synthesized with the common four ribonucleoside triphosphates (rNTPs) or rNTPs and deoxyribonucleoside triphosphates (dNTPs) in the reaction medium, were analyzed by polyacrylamide gel electrophoresis (PAGE) at denaturing conditions. It was shown that COMDP strongly activates the Pr and uncouples its activity from that of alpha DNA pol with accumulation of initiator (i) RNAs of the basic length. This phenomenon is not affected by BuPdGTP. MIMO, in contrast, stimulates both pol activities of this enzyme complex and preserves their mutual coupling. The effects of COMDP, MIMO and CPX seem to be modulated by concentration of the ambient dNTPs. Addition of dNTPs to rNTPs makes the effects of COMDP and MIMO mutually exclusive, suggesting that both these agents, though chemically quite different, are competing for one active site responsible for coupling these both pol activities into the one Pr-alpha DNA pol reaction.

Avian Myeloblastosis Virus↗
PubMed1994

The 50-kDa primase subunit of Drosophila melanogaster DNA polymerase alpha. Molecular characterization of the gene and functional analysis of the overexpressed protein.

The gene encoding the 50-kDa subunit of Drosophila melanogaster DNA polymerase alpha has been cloned. A comparison of the predicted polypeptide sequence of the Drosophila protein with the equivalent subunits from mouse and yeast suggests that they are closely related and defines three conserved regions which are likely to be important for enzyme activity. The expression patterns of both the 50-kDa protein and its transcript (a single RNA message of 1.6 kilobases) throughout development are consistent with a role of the protein in DNA replication. When overexpressed and purified the 50-kDa subunit displays DNA primase activity. The products of the reaction, mainly oligoribonucleotides 12-14 nucleotides in length, plus dimers and some trimers, are similar to those synthesized by either the intact DNA polymerase alpha, or the biochemically isolated primase heterodimer. The isolated primase also shows similar sensitivity to antibodies, magnesium and monovalent cations, and the same nucleotide requirements as complexed forms of the primase. The isolated subunit, however, is more thermally labile, suggesting a role for the additional subunits in DNA polymerase alpha in stabilizing the primase activity of the 50-kDa primase subunit.

Amino Acid Sequence↗
PubMed1987

Pre-replicative association of multiple replicative enzyme activities with the nuclear matrix during rat liver regeneration.

As a step toward the molecular elucidation of the putative replicational apparatus associated with the nuclear matrix, we have investigated the possible matrix association of several replicational related enzymes. In addition to the previously identified DNA polymerase alpha, DNA primase, 3'-5' exonuclease, RNase H, and DNA methylase were all recovered at significant levels (20-30% of total nuclear activity) in nuclear matrix isolated from regenerating rat liver during maximal in vivo replication (22 h post-hepatectomy). In contrast, DNA ligase was not detected on the nuclear matrix even though significant activity was present in isolated nuclei. Examination of the replicative dependency of these enzyme activities following partial hepatectomy revealed pre-replicative elevations which were distinct for each matrix-bound enzyme. A second late-replicative peak in DNA methylase is consistent with a role of this matrix-bound enzyme in the maintenance of the inheritable methylation pattern. Mild sonication resulted in a significant release of all of these activities except RNase H. A major portion of the matrix-solubilized DNA polymerase alpha, DNA primase, 3'-5' exonuclease, and DNA methylase activities cosedimented on sucrose gradients between approximately 8-12 S. Our results are consistent with the organization of at least a portion of these replicative enzymes into nuclear matrix-bound replicational complexes. We also propose a novel pre-replicative assembly model of the matrix-bound replicational apparatus in which DNA primase plays an initial and critical role.

Animals↗
PubMed2004

Structure of a bifunctional DNA primase-polymerase.

Genome replication generally requires primases, which synthesize an initial oligonucleotide primer, and DNA polymerases, which elongate the primer. Primase and DNA polymerase activities are combined, however, in newly identified replicases from archaeal plasmids, such as pRN1 from Sulfolobus islandicus. Here we present a structure-function analysis of the pRN1 primase-polymerase (prim-pol) domain. The crystal structure shows a central depression lined by conserved residues. Mutations on one side of the depression reduce DNA affinity. On the opposite side of the depression cluster three acidic residues and a histidine, which are required for primase and DNA polymerase activity. One acidic residue binds a manganese ion, suggestive of a metal-dependent catalytic mechanism. The structure does not show any similarity to DNA polymerases, but is distantly related to archaeal and eukaryotic primases, with corresponding active-site residues. We propose that archaeal and eukaryotic primases and the prim-pol domain have a common evolutionary ancestor, a bifunctional replicase for small DNA genomes.

Amino Acid Sequence↗