Plasmid RP4 specifies a deoxyribonucleic acid primase involved in its conjugal transfer and maintenance.

We surveyed plasmids representative of most incompatibility groups for their conferred deoxyribonucleic acid (DNA) primase activity. RP4 (IncP) was one of the few with such activity although, unlike the derepressed IncIalpha plasmids (which also specify a primase), it did not suppress the dnaG mutation. Using deletion and Tn7 derivatives of RP4, we located the presumed primase structural gene (pri) in the 37- to 42-kilobase region. Tn7 insertions in the adjacent Tra1 region also reduced or caused overproduction of primase. We purified the RP4 primase to a single polypeptide of molecular weight 118,000. It is an anisometric molecule and functions as a monomer, initiating complementary strand synthesis on phi X174 DNA in Escherichia coli dnaG cell extracts in the presence of ribonucleotide triphosphates and rifampin. It is immunologically unrelated to either the E. coli dnaG or the IncIalpha plasmid-specified DNA primases. RP4 pri mutants conjugated with a lower efficiency into some bacterial species, including Salmonella typhimurium. Back-transfer experiments showed that this effect was recipient specific. There was also a comparable reduction in mobilization efficiency of R300B by RP4 pri into such recipients. Loss of RP4 primase led to detectable plasmid instability. The RP4-specified primase therefore seems to serve two functions: the single DNA strand transferred during conjugation is primed by it in the recipient cell, and it appears to be necessary for the efficient priming of discontinuous plasmid DNA replication despite the presence of the chromosomal priming system.     

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

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.     

Bacillus subtilis dnaE encodes a protein homologous to DNA primase of Escherichia coli

Bacillus subtilis dnaE encodes a protein essential for DNA replication and is tightly linked to rpoD, the gene for the major sigma factor of RNA polymerase. We have now determined the 1809-base pair sequence of the dnaE coding region, which precedes rpoD and is transcribed in the same counterclockwise direction on the chromosome. From the DNA sequence, we found that the dnaE protein comprised 603 amino acids with a calculated molecular mass of 68,428 daltons. This protein had significant and extensive regions of homology with Escherichia coli DNA primase, the polymerase that synthesizes short RNA primers during discontinuous DNA replication. Features of the coding and flanking regions that may modulate dnaE expression include a relatively weak ribosomal binding site (delta G' = -13.8 kcal), the use of uncommon codons in the reading frame, and no obvious promoter sequence for either dnaE or rpoD. Together, these results suggest that dnaE codes for B. subtilis DNA primase and, in light of the similarities to the organization of the E. coli sigma operon, that expression of dnaE may be coregulated with rpoD in B. subtilis.     

Plasmid RP4, with Escherichia coli DNA inserted in vitro, mediates chromosomal transfer.

The wide host-range plasmid RP4 is generally poor at mobilizing chromosomal markers. Insertion in vitro of Escherichia coli chromosomal DNA into its HindIII site (recognized by loss of its kanamycin resistance marker) or its EcoRI site generated RP4-primes that efficiently mobilized chromosomal markers. These RP4-primes behaved analogously to F-primes, each had a particular transfer origin and direction of marker mobilization, and this was abolished by a recA allele in the donor. Insertion sequences on the RP4-primes do not appear to be necessary for mobilization, it seems very unlikely that each of the RP4-primes generated contained an insertion sequence. The construction and use of such plasmids should facilitate inter- and intrageneric genetic analyses and manipulations.     

Genetic Rearrangement of Plasmids: In Vivo Recombination between a Dehalogenation Plasmid and Multiple-Resistance Plasmid RP4 in Pseudomonas sp

When Moraxella plasmid pUO1 encoding haloacetate dehalogenase and mercury resistance coexisted with IncP-1 plasmid RP4 in Pseudomonas sp., genetic exchange between the plasmids often occurred, probably by site-specific recombination. The recombinant plasmids obtained were classified into four groups on the basis of phenotype. Representative plasmids for each group were analyzed for DNA composition and function, and the mechanism for the formation of these plasmids was sought. They were inherited stably in Escherichia coli and a Pseudomonas sp.     

Accuracy of DNA primase

The base-pairing fidelity of DNA primase from calf thymus was studied in vitro by using a misinsertion assay based on synthetic polydeoxynucleotide templates. With poly(dT) as template, GMP misinsertions occurred with a frequency of one error per 1600 correctly incorporated nucleotides, while UMP and CMP were inserted with a frequency of 1/300 and 1/500, respectively. Accuracy with poly(dC, dT) as template was 1/200 for the misinsertion of UMP, and 1/300 for the misinsertion of CMP. Thus, DNA primase is the least accurate polynucleotide-synthesizing enzyme known. The results are discussed in terms of an obvious necessity for a priming mechanism at the beginning of DNA synthesis.     

Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.     

Site-Specific Mutations in the TraI Relaxase and Upstream Region of Plasmid RP4

The relaxase of RP4 nicks the double-stranded plasmid at the oriT site and binds covalently to DNA at the 5′ end of the nick. The 80-kDa relaxase (TraI) is encoded on an operon with several overlapping open reading frames (ORFs). The importance in conjugation of a short ORF (traX) with a start site overlapping the 5′ terminus of traI was investigated, as well as the effects of specific mutations in the relaxase. Elimination of TraX reduced the transfer efficiency by approximately 50% in several intergeneric matings, especially when Escherichia coli was the donor. While TraI was essential for transfer to occur, deletion of the C-terminus of TraI decreased, but did not eliminate plasmid transfer. Mutation of the active site tyrosine resulted in residual transfer associated with amino acid misincorporation.     

DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha

A very highly purified fraction of KB cell DNA polymerase-alpha, prepared with a monoclonal antibody, contains DNA primase activity. The primase synthesizes oligonucleotide chains initiated with ATP in a reaction that is resistant to alpha-amanitin and strictly dependent on added template and ribonucleoside triphosphates (rNTPs). In the presence of added dNTPs and M13 DNA template, the primase produces a uniform population of oligoribonucleotides, predominantly hexamers to decamers, that are extended by polymerase-alpha into DNA chains up to 3000 nucleotides long. There is no evidence for nucleotide preferences at RNA/DNA junctions. In the absence of added dNTPs, the oligomeric products are heterogeneous in size and composition and susceptible to cleavage by pancreatic DNase I due to their content of short oligodeoxynucleotide tracts synthesized by primase from trace contaminant dNTPs in the rNTP substrates. The primase and polymerase-alpha activities are distinguishable by several physical and chemical criteria, and the primase reaction is only partially sensitive to two potent, independent monoclonal antibodies that neutralize polymerase-alpha. Although the presence of both primase and polymerase-alpha activities in a highly purified immune complex prepared with a monoclonal antibody argues for their tight physical association, the chemical, physical, and immunological discriminations indicate the two catalytic entities are functionally and structurally distinct.     

Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex

An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

Purification and properties of a DNA primase from Nicotianatabacum

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase α-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase. Received: 8 May 1997 / Accepted: 5 June 1997     

Characterization of a DNA primase from rat liver mitochondria

A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.     

Cell-cycle-dependent expression of DNA primase activity

Protein extracts were prepared at various times after serum stimulation of growth-arrested mouse 3T3 fibroblasts. The extracts were fractionated by sucrose gradient centrifugation and used to determine the activities of DNA polymerase alpha and DNA primase. We found that polymerase and primase appeared in close association in one homogeneous 8.2-S peak. Neither polymerase, free of associated primase, nor primase, free of polymerase, could be detected at any time after serum stimulation. The activities of both enzymes started to increase concomitantly at the beginning of the DNA replication phase of the cell cycle. We found five to six times more DNA primase activity in replicating than in resting 3T3 cells. Besides DNA primase, a second additional priming activity could be detected. This activity sedimented at 12.5 S and corresponded most probably to RNA polymerase I.     

Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity.

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.