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. 1984 Oct;81(20):6456–6460. doi: 10.1073/pnas.81.20.6456

Plasmid P1 replication: negative control by repeated DNA sequences.

D Chattoraj, K Cordes, A Abeles
PMCID: PMC391943  PMID: 6387706

Abstract

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro.

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Selected References

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

  1. Abeles A. L., Snyder K. M., Chattoraj D. K. P1 plasmid replication: replicon structure. J Mol Biol. 1984 Mar 5;173(3):307–324. doi: 10.1016/0022-2836(84)90123-2. [DOI] [PubMed] [Google Scholar]
  2. Alton N. K., Vapnek D. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature. 1979 Dec 20;282(5741):864–869. doi: 10.1038/282864a0. [DOI] [PubMed] [Google Scholar]
  3. Austin S., Abeles A. Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition. J Mol Biol. 1983 Sep 15;169(2):353–372. doi: 10.1016/s0022-2836(83)80055-2. [DOI] [PubMed] [Google Scholar]
  4. Austin S., Hart F., Abeles A., Sternberg N. Genetic and physical map of a P1 miniplasmid. J Bacteriol. 1982 Oct;152(1):63–71. doi: 10.1128/jb.152.1.63-71.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  6. Beckingham K. A plasmid cloning vector for Kpnl-cleaved DNA. Plasmid. 1980 Nov;4(3):354–356. doi: 10.1016/0147-619x(80)90074-8. [DOI] [PubMed] [Google Scholar]
  7. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  8. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Froehlich B. J., Tatti K., Scott J. R. Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants. J Bacteriol. 1983 Oct;156(1):205–211. doi: 10.1128/jb.156.1.205-211.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Kamio Y., Terawaki Y. Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats. J Bacteriol. 1983 Sep;155(3):1185–1191. doi: 10.1128/jb.155.3.1185-1191.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maki S., Miki T., Horiuchi T. DNA sequence of an amber replication mutant indicates that a 29 kd protein is the product of the F plasmid replication gene. Mol Gen Genet. 1984;194(1-2):337–339. doi: 10.1007/BF00383537. [DOI] [PubMed] [Google Scholar]
  14. Mizusawa S., Ward D. F. A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene. 1982 Dec;20(3):317–322. doi: 10.1016/0378-1119(82)90200-1. [DOI] [PubMed] [Google Scholar]
  15. Prentki P., Chandler M., Caro L. Replication of prophage P1 during the cell cycle of Escherichia coli. Mol Gen Genet. 1977 Mar 28;152(1):71–76. doi: 10.1007/BF00264942. [DOI] [PubMed] [Google Scholar]
  16. Rosner J. L. Formation, induction, and curing of bacteriophage P1 lysogens. Virology. 1972 Jun;48(3):679–689. doi: 10.1016/0042-6822(72)90152-3. [DOI] [PubMed] [Google Scholar]
  17. Seelke R. W., Kline B. C., Trawick J. D., Ritts G. D. Genetic studies of F plasmid maintenance genes involved in copy number control, incompatability, and partitioning. Plasmid. 1982 Mar;7(2):163–179. doi: 10.1016/0147-619x(82)90075-0. [DOI] [PubMed] [Google Scholar]
  18. Smith G. R. Deletion mutations of the immunity region of coliphage lambda. Virology. 1975 Apr;64(2):544–552. doi: 10.1016/0042-6822(75)90131-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Sternberg N., Hamilton D., Hoess R. Bacteriophage P1 site-specific recombination. II. Recombination between loxP and the bacterial chromosome. J Mol Biol. 1981 Aug 25;150(4):487–507. doi: 10.1016/0022-2836(81)90376-4. [DOI] [PubMed] [Google Scholar]
  22. Sternberg N., Powers M., Yarmolinsky M., Austin S. Group Y incompatibility and copy control of P1 prophage. Plasmid. 1981 Mar;5(2):138–149. doi: 10.1016/0147-619x(81)90015-9. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Tsutsui H., Fujiyama A., Murotsu T., Matsubara K. Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control. J Bacteriol. 1983 Jul;155(1):337–344. doi: 10.1128/jb.155.1.337-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Vocke C., Bastia D. Primary structure of the essential replicon of the plasmid pSC101. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6557–6561. doi: 10.1073/pnas.80.21.6557. [DOI] [PMC free article] [PubMed] [Google Scholar]

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