Abstract
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.
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Selected References
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- Arai K., Low R. L., Kornberg A. Movement and site selection for priming by the primosome in phage phi X174 DNA replication. Proc Natl Acad Sci U S A. 1981 Feb;78(2):707–711. doi: 10.1073/pnas.78.2.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barth P. T., Grinter N. J., Bradley D. E. Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion. J Bacteriol. 1978 Jan;133(1):43–52. doi: 10.1128/jb.133.1.43-52.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barth P. T., Grinter N. J. Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides. J Bacteriol. 1974 Nov;120(2):618–630. doi: 10.1128/jb.120.2.618-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barth P. T., Grinter N. J. Map of plasmid RP4 derived by insertion of transposon C. J Mol Biol. 1977 Jul 5;113(3):455–474. doi: 10.1016/0022-2836(77)90233-9. [DOI] [PubMed] [Google Scholar]
- Boulnois G. J., Wilkins B. M. A novel priming system for conjugal synthesis of an IncI alpha plasmid in recipients. Mol Gen Genet. 1979 Oct 1;175(3):275–279. doi: 10.1007/BF00397227. [DOI] [PubMed] [Google Scholar]
- Bradley D. E., Taylor D. E., Cohen D. R. Specification of surface mating systems among conjugative drug resistance plasmids in Escherichia coli K-12. J Bacteriol. 1980 Sep;143(3):1466–1470. doi: 10.1128/jb.143.3.1466-1470.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Godson G. N., Roberts R. J. dna, single stranded/*metab. Virology. 1976 Sep;73(2):561–567. doi: 10.1016/0042-6822(76)90421-9. [DOI] [PubMed] [Google Scholar]
- Guiney D. G., Helinski D. R. The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer. Mol Gen Genet. 1979 Oct 3;176(2):183–189. doi: 10.1007/BF00273212. [DOI] [PubMed] [Google Scholar]
- Lanka E., Mikolajczyk M., Schlicht M., Schuster H. Association of the prophage P1ban protein with the dnaB protein of Escherichia coli. J Biol Chem. 1978 Jul 10;253(13):4746–4753. [PubMed] [Google Scholar]
- Lanka E., Scherzinger E., Günther E., Schuster H. A DNA primase specified by I-like plasmids. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3632–3636. doi: 10.1073/pnas.76.8.3632. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lanka E., Schuster H. Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis. Mol Gen Genet. 1970;106(3):274–285. doi: 10.1007/BF00340386. [DOI] [PubMed] [Google Scholar]
- Ludwig R. A., Johansen E. DnaG-suppressing variants of R68.45 with enhanced chromosome donating ability in Rhizobium. Plasmid. 1980 May;3(3):359–361. doi: 10.1016/0147-619x(80)90049-9. [DOI] [PubMed] [Google Scholar]
- Nordheim A., Hashimoto-Gotoh T., Timmis K. N. Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid. J Bacteriol. 1980 Dec;144(3):923–932. doi: 10.1128/jb.144.3.923-932.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasakawa C., Yoshikawa M. Requirements for suppression of a dnaG mutation by an I-type plasmid. J Bacteriol. 1978 Feb;133(2):485–491. doi: 10.1128/jb.133.2.485-491.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schekman R., Weiner A., Kornberg A. Multienzyme systems of DNA replication. Science. 1974 Dec 13;186(4168):987–993. doi: 10.1126/science.186.4168.987. [DOI] [PubMed] [Google Scholar]
- Schuster H., Schlicht M., Lanka E., Mikolajczyk M., Edelbluth C. DNA synthesis in an Escherichia coli dna B dnaC mutant. Mol Gen Genet. 1977 Feb 28;151(1):11–16. doi: 10.1007/BF00446907. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vapnek D., Lipman M. B., Rupp W. D. Physical properties and mechanism of transfer of R factors in Escherichia coli. J Bacteriol. 1971 Oct;108(1):508–514. doi: 10.1128/jb.108.1.508-514.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vapnek D., Rupp W. D. Asymmetric segregation of the complementary sex-factor DNA strands during conjugation in Escherichia coli. J Mol Biol. 1970 Nov 14;53(3):287–303. doi: 10.1016/0022-2836(70)90066-5. [DOI] [PubMed] [Google Scholar]
- Wechsler J. A., Nüsslein V., Otto B., Klein A., Bonhoeffer F., Herrmann R., Gloger L., Schaller H. Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication. J Bacteriol. 1973 Mar;113(3):1381–1388. doi: 10.1128/jb.113.3.1381-1388.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wickner S., Wright M., Hurwitz J. Studies on in vitro DNA synthesis. Purification of the dna G gene product from Escherichia coli. Proc Natl Acad Sci U S A. 1973 May;70(5):1613–1618. doi: 10.1073/pnas.70.5.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilkins B. M., Boulnois G. J., Lanka E. A plasmid DNA primase active in discontinuous bacterial DNA replication. Nature. 1981 Mar 19;290(5803):217–221. doi: 10.1038/290217a0. [DOI] [PubMed] [Google Scholar]
- Wilkins B. M. Partial suppression of the phenotype of Escherichia coli K-12 dnaG mutants by some I-like conjugative plasmids. J Bacteriol. 1975 Jun;122(3):899–904. doi: 10.1128/jb.122.3.899-904.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Windass J. D., Worsey M. J., Pioli E. M., Pioli D., Barth P. T., Atherton K. T., Dart E. C., Byrom D., Powell K., Senior P. J. Improved conversion of methanol to single-cell protein by Methylophilus methylotrophus. Nature. 1980 Oct 2;287(5781):396–401. doi: 10.1038/287396a0. [DOI] [PubMed] [Google Scholar]
- Wright M., Wickner S., Hurwitz J. Studies on in vitro DNA synthesis. Isolation of DNA B gene product from Escherichia coli. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3120–3124. doi: 10.1073/pnas.70.11.3120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zyskind J. W., Smith D. W. NOVEL Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiaion of a round of deoxyribonucleic acid replication. J Bacteriol. 1977 Mar;129(3):1476–1486. doi: 10.1128/jb.129.3.1476-1486.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]