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. 1986 Aug;83(15):5484–5488. doi: 10.1073/pnas.83.15.5484

Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication.

R R Koepsel, R W Murray, S A Khan
PMCID: PMC386311  PMID: 3461445

Abstract

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.

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Selected References

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

  1. Braun R. E., O'Day K., Wright A. Autoregulation of the DNA replication gene dnaA in E. coli K-12. Cell. 1985 Jan;40(1):159–169. doi: 10.1016/0092-8674(85)90319-8. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chattoraj D. K., Snyder K. M., Abeles A. L. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc Natl Acad Sci U S A. 1985 May;82(9):2588–2592. doi: 10.1073/pnas.82.9.2588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Galas D. J., Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978 Sep;5(9):3157–3170. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Germino J., Bastia D. Interaction of the plasmid R6K-encoded replication initiator protein with its binding sites on DNA. Cell. 1983 Aug;34(1):125–134. doi: 10.1016/0092-8674(83)90142-3. [DOI] [PubMed] [Google Scholar]
  7. Germino J., Bastia D. The replication initiator protein of plasmid R6K tagged with beta-galactosidase shows sequence-specific DNA-binding. Cell. 1983 Jan;32(1):131–140. doi: 10.1016/0092-8674(83)90503-2. [DOI] [PubMed] [Google Scholar]
  8. Kaguni J. M., Kornberg A. Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes. Cell. 1984 Aug;38(1):183–190. doi: 10.1016/0092-8674(84)90539-7. [DOI] [PubMed] [Google Scholar]
  9. Kelley W., Bastia D. Replication initiator protein of plasmid R6K autoregulates its own synthesis at the transcriptional step. Proc Natl Acad Sci U S A. 1985 May;82(9):2574–2578. doi: 10.1073/pnas.82.9.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Koepsel R. R., Murray R. W., Rosenblum W. D., Khan S. A. The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6845–6849. doi: 10.1073/pnas.82.20.6845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. McEachern M. J., Filutowicz M., Helinski D. R. Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1480–1484. doi: 10.1073/pnas.82.5.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Murotsu T., Matsubara K., Sugisaki H., Takanami M. Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene. 1981 Nov;15(2-3):257–271. doi: 10.1016/0378-1119(81)90135-9. [DOI] [PubMed] [Google Scholar]
  19. Murotsu T., Tsutsui H., Matsubara K. Identification of the minimal essential region for the replication origin of miniF plasmid. Mol Gen Genet. 1984;196(2):373–378. doi: 10.1007/BF00328075. [DOI] [PubMed] [Google Scholar]
  20. Nordheim A., Lafer E. M., Peck L. J., Wang J. C., Stollar B. D., Rich A. Negatively supercoiled plasmids contain left-handed Z-DNA segments as detected by specific antibody binding. Cell. 1982 Dec;31(2 Pt 1):309–318. doi: 10.1016/0092-8674(82)90124-6. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Ogata R. T., Gilbert W. An amino-terminal fragment of lac repressor binds specifically to lac operator. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5851–5854. doi: 10.1073/pnas.75.12.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rich A. Right-handed and left-handed DNA: conformational information in genetic material. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):1–12. doi: 10.1101/sqb.1983.047.01.003. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Shafferman A., Kolter R., Stalker D., Helinski D. R. Plasmid R6K DNA replication. III. Regulatory properties of the pi initiation protein. J Mol Biol. 1982 Oct 15;161(1):57–76. doi: 10.1016/0022-2836(82)90278-9. [DOI] [PubMed] [Google Scholar]
  28. Stalker D. M., Kolter R., Helinski D. R. Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1150–1154. doi: 10.1073/pnas.76.3.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  30. Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. doi: 10.1016/0092-8674(78)90147-2. [DOI] [PubMed] [Google Scholar]
  31. Tsurimoto T., Matsubara K. Purified bacteriophage lambda O protein binds to four repeating sequences at the lambda replication origin. Nucleic Acids Res. 1981 Apr 24;9(8):1789–1799. doi: 10.1093/nar/9.8.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vocke C., Bastia D. DNA-protein interaction at the origin of DNA replication of the plasmid pSC101. Cell. 1983 Dec;35(2 Pt 1):495–502. doi: 10.1016/0092-8674(83)90183-6. [DOI] [PubMed] [Google Scholar]
  33. Vocke C., Bastia D. The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2252–2256. doi: 10.1073/pnas.82.8.2252. [DOI] [PMC free article] [PubMed] [Google Scholar]

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