Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1991 Mar 25;19(6):1203–1211. doi: 10.1093/nar/19.6.1203

Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins.

E Scherzinger 1, V Haring 1, R Lurz 1, S Otto 1
PMCID: PMC333844  PMID: 1851552

Abstract

We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-pair origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (gamma subunit of poIIII holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the replicative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.

Full text

PDF
1205

Images in this article

1205Image
on p.1205
1208Image
on p.1208

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alfano C., McMacken R. The role of template superhelicity in the initiation of bacteriophage lambda DNA replication. Nucleic Acids Res. 1988 Oct 25;16(20):9611–9630. doi: 10.1093/nar/16.20.9611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bazaral M., Helinski D. R. Replication of a bacterial plasmid and an episome in Escherichia coli. Biochemistry. 1970 Jan 20;9(2):399–406. doi: 10.1021/bi00804a029. [DOI] [PubMed] [Google Scholar]
  4. Bramhill D., Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 1988 Mar 11;52(5):743–755. doi: 10.1016/0092-8674(88)90412-6. [DOI] [PubMed] [Google Scholar]
  5. Böldicke T. W., Hillenbrand G., Lanka E., Staudenbauer W. L. Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA. Nucleic Acids Res. 1981 Oct 24;9(20):5215–5231. doi: 10.1093/nar/9.20.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carl P. L. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol Gen Genet. 1970;109(2):107–122. doi: 10.1007/BF00269647. [DOI] [PubMed] [Google Scholar]
  7. Chase J. W., Whittier R. F., Auerbach J., Sancar A., Rupp W. D. Amplification of single-strand DNA binding protein in Escherichia coli. Nucleic Acids Res. 1980 Jul 25;8(14):3215–3227. doi: 10.1093/nar/8.14.3215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen H., Lawrence C. B., Bryan S. K., Moses R. E. Aphidicolin inhibits DNA polymerase II of Escherichia coli, an alpha-like DNA polymerase. Nucleic Acids Res. 1990 Dec 11;18(23):7185–7186. doi: 10.1093/nar/18.23.7185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Derbyshire K. M., Hatfull G., Willetts N. Mobilization of the non-conjugative plasmid RSF1010: a genetic and DNA sequence analysis of the mobilization region. Mol Gen Genet. 1987 Jan;206(1):161–168. doi: 10.1007/BF00326552. [DOI] [PubMed] [Google Scholar]
  10. Derbyshire K. M., Willetts N. S. Mobilization of the non-conjugative plasmid RSF1010: a genetic analysis of its origin of transfer. Mol Gen Genet. 1987 Jan;206(1):154–160. doi: 10.1007/BF00326551. [DOI] [PubMed] [Google Scholar]
  11. Diaz R., Staudenbauer W. L. Replication of the broad host range plasmid RSF1010 in cell-free extracts of Escherichia coli and Pseudomonas aeruginosa. Nucleic Acids Res. 1982 Aug 11;10(15):4687–4702. doi: 10.1093/nar/10.15.4687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Filip C. C., Allen J. S., Gustafson R. A., Allen R. G., Walker J. R. Bacterial cell division regulation: characterization of the dnaH locus of Escherichia coli. J Bacteriol. 1974 Aug;119(2):443–449. doi: 10.1128/jb.119.2.443-449.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuller R. S., Funnell B. E., Kornberg A. The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 1984 Oct;38(3):889–900. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  14. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  15. Guerry P., van Embden J., Falkow S. Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J Bacteriol. 1974 Feb;117(2):619–630. doi: 10.1128/jb.117.2.619-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haring V., Scholz P., Scherzinger E., Frey J., Derbyshire K., Hatfull G., Willetts N. S., Bagdasarian M. Protein RepC is involved in copy number control of the broad host range plasmid RSF1010. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6090–6094. doi: 10.1073/pnas.82.18.6090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  18. Honda Y., Sakai H., Komano T., Bagdasarian M. RepB' is required in trans for the two single-strand DNA initiation signals in oriV of plasmid RSF1010. Gene. 1989 Aug 1;80(1):155–159. doi: 10.1016/0378-1119(89)90261-8. [DOI] [PubMed] [Google Scholar]
  19. Hübscher U., Kornberg A. The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1980 Dec 25;255(24):11698–11703. [PubMed] [Google Scholar]
  20. Kaguni J. M., Fuller R. S., Kornberg A. Enzymatic replication of E. coli chromosomal origin is bidirectional. Nature. 1982 Apr 15;296(5858):623–627. doi: 10.1038/296623a0. [DOI] [PubMed] [Google Scholar]
  21. Lanka E., Schuster H. The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein. Nucleic Acids Res. 1983 Feb 25;11(4):987–997. doi: 10.1093/nar/11.4.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Masai H., Arai K. Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT. J Biol Chem. 1988 Oct 15;263(29):15016–15023. [PubMed] [Google Scholar]
  23. Masai H., Nomura N., Kubota Y., Arai K. Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication. J Biol Chem. 1990 Sep 5;265(25):15124–15133. [PubMed] [Google Scholar]
  24. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  25. Morris C. F., Sinha N. K., Alberts B. M. Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4800–4804. doi: 10.1073/pnas.72.12.4800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ortega S., Lanka E., Diaz R. The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA. Nucleic Acids Res. 1986 Jun 25;14(12):4865–4879. doi: 10.1093/nar/14.12.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Scherzinger E., Bagdasarian M. M., Scholz P., Lurz R., Rückert B., Bagdasarian M. Replication of the broad host range plasmid RSF1010: requirement for three plasmid-encoded proteins. Proc Natl Acad Sci U S A. 1984 Feb;81(3):654–658. doi: 10.1073/pnas.81.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scholz P., Haring V., Scherzinger E., Lurz R., Bagdasarian M. M., Schuster H., Bagdasarian M. Replication determinants of the broad host-range plasmid RSF1010. Basic Life Sci. 1985;30:243–259. doi: 10.1007/978-1-4613-2447-8_20. [DOI] [PubMed] [Google Scholar]
  29. Scholz P., Haring V., Wittmann-Liebold B., Ashman K., Bagdasarian M., Scherzinger E. Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010. Gene. 1989 Feb 20;75(2):271–288. doi: 10.1016/0378-1119(89)90273-4. [DOI] [PubMed] [Google Scholar]
  30. Schuster H., Mikolajczyk M., Rohrschneider J., Geschke B. phiX174 DNA-dependent DNA synthesis in vitro: requirement for P1 ban protein in dnaB mutant extracts of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3907–3911. doi: 10.1073/pnas.72.10.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Staudenbauer W. L., Lanka E., Schuster H. Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates. Mol Gen Genet. 1978 Jul 4;162(3):243–249. doi: 10.1007/BF00268849. [DOI] [PubMed] [Google Scholar]
  32. Staudenbauer W. L. Replication of small plasmids in extracts of Escherichia coli. Mol Gen Genet. 1976 Jun 15;145(3):273–280. doi: 10.1007/BF00325823. [DOI] [PubMed] [Google Scholar]
  33. Staudenbauer W. L. Replication of the ampicillin resistance plasmid RSF1030 in extracts of Escherichia coli: separation of the replication cycle into early and late stages. Mol Gen Genet. 1977 Nov 4;156(1):27–34. doi: 10.1007/BF00272248. [DOI] [PubMed] [Google Scholar]
  34. Tippe-Schindler R., Zahn G., Messer W. Control of the initiation of DNA replication in Escherichia coli. I. Negative control of initiation. Mol Gen Genet. 1979 Jan 10;168(2):185–195. doi: 10.1007/BF00431444. [DOI] [PubMed] [Google Scholar]
  35. Uhlin B. E., Nordström K. A runaway-replication mutant of plasmid R1drd-19: temperature-dependent loss of copy number control. Mol Gen Genet. 1978 Oct 4;165(2):167–179. doi: 10.1007/BF00269904. [DOI] [PubMed] [Google Scholar]
  36. Waddell J., Wang X. M., Wu M. Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardii. Nucleic Acids Res. 1984 May 11;12(9):3843–3856. doi: 10.1093/nar/12.9.3843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wickner S., Hurwitz J. Involvement of escherichia coli dnaZ gene product in DNA elongation in vitro. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1053–1057. doi: 10.1073/pnas.73.4.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Younghusband H. B., Inman R. B. The electronmicroscopy of DNA. Annu Rev Biochem. 1974;43(0):605–619. doi: 10.1146/annurev.bi.43.070174.003133. [DOI] [PubMed] [Google Scholar]
  39. de Graaff J., Crosa J. H., Heffron F., Falkow S. Replication of the nonconjugative plasmid RSF1010 in Escherichia coli K-12. J Bacteriol. 1978 Jun;134(3):1117–1122. doi: 10.1128/jb.134.3.1117-1122.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES