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. 1985 Oct;82(20):6845–6849. doi: 10.1073/pnas.82.20.6845

The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities.

R R Koepsel, R W Murray, W D Rosenblum, S A Khan
PMCID: PMC390784  PMID: 2995991

Abstract

Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequence-specific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein. RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT181 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

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Selected References

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

  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Carleton S., Projan S. J., Highlander S. K., Moghazeh S. M., Novick R. P. Control of pT181 replication II. Mutational analysis. EMBO J. 1984 Oct;3(10):2407–2414. doi: 10.1002/j.1460-2075.1984.tb02147.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conrad S. E., Campbell J. L. Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids. Nucleic Acids Res. 1979 Jul 25;6(10):3289–3304. doi: 10.1093/nar/6.10.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Diaz R., Nordström K., Staudenbauer W. L. Plasmid R1 DNA replication dependent on protein synthesis in cell-free extracts of E. coli. Nature. 1981 Jan 22;289(5795):326–328. doi: 10.1038/289326a0. [DOI] [PubMed] [Google Scholar]
  8. Dotto G. P., Horiuchi K., Zinder N. D. Initiation and termination of phage f1 plus-strand synthesis. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7122–7126. doi: 10.1073/pnas.79.23.7122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eisenberg S., Griffith J., Kornberg A. phiX174 cistron A protein is a multifunctional enzyme in DNA replication. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3198–3202. doi: 10.1073/pnas.74.8.3198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eisenberg S., Kornberg A. Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication. J Biol Chem. 1979 Jun 25;254(12):5328–5332. [PubMed] [Google Scholar]
  11. Fuller R. S., Kaguni J. M., Kornberg A. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7370–7374. doi: 10.1073/pnas.78.12.7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  13. Germino J., Gray J. G., Charbonneau H., Vanaman T., Bastia D. Use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid R6K. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6848–6852. doi: 10.1073/pnas.80.22.6848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilbert W., Dressler D. DNA replication: the rolling circle model. Cold Spring Harb Symp Quant Biol. 1968;33:473–484. doi: 10.1101/sqb.1968.033.01.055. [DOI] [PubMed] [Google Scholar]
  15. Ikeda J. E., Yudelevich A., Hurwitz J. Isolation and characterization of the protein coded by gene A of bacteriophage phiX174 DNA. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2669–2673. doi: 10.1073/pnas.73.8.2669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Inuzuka M., Helinski D. R. Requirement of a plasmid-encoded protein for replication in vitro of plasmid R6K. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5381–5385. doi: 10.1073/pnas.75.11.5381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iordănescu S. Three distinct plasmids originating in the same Staphylococcus aureus strain. Arch Roum Pathol Exp Microbiol. 1976 Jan-Jun;35(1-2):111–118. [PubMed] [Google Scholar]
  18. Itoh T., Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. doi: 10.1073/pnas.77.5.2450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khan S. A., Adler G. K., Novick R. P. Functional origin of replication of pT181 plasmid DNA is contained within a 168-base-pair segment. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4580–4584. doi: 10.1073/pnas.79.15.4580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Khan S. A., Carleton S. M., Novick R. P. Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4902–4906. doi: 10.1073/pnas.78.8.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Khan S. A., Novick R. P. Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Plasmid. 1983 Nov;10(3):251–259. doi: 10.1016/0147-619x(83)90039-2. [DOI] [PubMed] [Google Scholar]
  22. Khan S. A., Novick R. P. Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production. J Bacteriol. 1982 Feb;149(2):642–649. doi: 10.1128/jb.149.2.642-649.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koepsel R. R., Murray R. W., Rosenblum W. D., Khan S. A. Purification of pT181-encoded repC protein required for the initiation of plasmid replication. J Biol Chem. 1985 Jul 15;260(14):8571–8577. [PubMed] [Google Scholar]
  24. Kumar C. C., Novick R. P. Plasmid pT181 replication is regulated by two countertranscripts. Proc Natl Acad Sci U S A. 1985 Feb;82(3):638–642. doi: 10.1073/pnas.82.3.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Langeveld S. A., van Mansfeld A. D., Baas P. D., Jansz H. S., van Arkel G. A., Weisbeek P. J. Nucleotide sequence of the origin of replication in bacteriophage phiX174 RF DNA. Nature. 1978 Feb 2;271(5644):417–420. doi: 10.1038/271417a0. [DOI] [PubMed] [Google Scholar]
  26. Liu L. F., Liu C. C., Alberts B. M. T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication. Nature. 1979 Oct 11;281(5731):456–461. doi: 10.1038/281456a0. [DOI] [PubMed] [Google Scholar]
  27. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  28. Meyer T. F., Geider K. Bacteriophage fd gene II-protein. I. Purification, involvement in RF replication, and the expression of gene II. J Biol Chem. 1979 Dec 25;254(24):12636–12641. [PubMed] [Google Scholar]
  29. Meyer T. F., Geider K. Bacteriophage fd gene II-protein. II. Specific cleavage and relaxation of supercoiled RF from filamentous phages. J Biol Chem. 1979 Dec 25;254(24):12642–12646. [PubMed] [Google Scholar]
  30. Meyer T. F., Geider K. Enzymatic synthesis of bacteriophage fd viral DNA. Nature. 1982 Apr 29;296(5860):828–832. doi: 10.1038/296828a0. [DOI] [PubMed] [Google Scholar]
  31. Meyer T. F., Geider K., Kurz C., Schaller H. Cleavage site of bacteriophage fd gene II-protein in the origin of viral strand replication. Nature. 1979 Mar 22;278(5702):365–367. doi: 10.1038/278365a0. [DOI] [PubMed] [Google Scholar]
  32. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  33. Novick R. P., Adler G. K., Majumder S., Khan S. A., Carleton S., Rosenblum W. D., Iordanescu S. Coding sequence for the pT181 repC product: a plasmid-coded protein uniquely required for replication. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4108–4112. doi: 10.1073/pnas.79.13.4108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Novick R. P., Adler G. K., Projan S. J., Carleton S., Highlander S. K., Gruss A., Khan S. A., Iordanescu S. Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein. EMBO J. 1984 Oct;3(10):2399–2405. doi: 10.1002/j.1460-2075.1984.tb02146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967 Sep;33(1):155–166. doi: 10.1016/0042-6822(67)90105-5. [DOI] [PubMed] [Google Scholar]
  36. Projan S. J., Carleton S., Novick R. P. Determination of plasmid copy number by fluorescence densitometry. Plasmid. 1983 Mar;9(2):182–190. doi: 10.1016/0147-619x(83)90019-7. [DOI] [PubMed] [Google Scholar]
  37. Scott J. R. Regulation of plasmid replication. Microbiol Rev. 1984 Mar;48(1):1–23. doi: 10.1016/b978-0-12-048850-6.50006-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Tjian R., Robbins A. Enzymatic activities associated with a purified simian virus 40 T antigen-related protein. Proc Natl Acad Sci U S A. 1979 Feb;76(2):610–614. doi: 10.1073/pnas.76.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsurimoto T., Matsubara K. Replication of bacteriophage lambda DNA. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):681–691. doi: 10.1101/sqb.1983.047.01.079. [DOI] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]

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