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. 1991 Oct 15;88(20):9011–9015. doi: 10.1073/pnas.88.20.9011

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

A L Abeles 1, S J Austin 1
PMCID: PMC52641  PMID: 1924364

Abstract

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

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Selected References

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  1. Abeles A. L., Austin S. J. P1 plasmid replication requires methylated DNA. EMBO J. 1987 Oct;6(10):3185–3189. doi: 10.1002/j.1460-2075.1987.tb02630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abeles A. L., Friedman S. A., Austin S. J. Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region. J Mol Biol. 1985 Sep 20;185(2):261–272. doi: 10.1016/0022-2836(85)90402-4. [DOI] [PubMed] [Google Scholar]
  3. Abeles A. L. P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA. J Biol Chem. 1986 Mar 15;261(8):3548–3555. [PubMed] [Google Scholar]
  4. Abeles A. L., Reaves L. D., Austin S. J. A single DnaA box is sufficient for initiation from the P1 plasmid origin. J Bacteriol. 1990 Aug;172(8):4386–4391. doi: 10.1128/jb.172.8.4386-4391.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Austin S. J., Mural R. J., Chattoraj D. K., Abeles A. L. Trans- and cis-acting elements for the replication of P1 miniplasmids. J Mol Biol. 1985 May 25;183(2):195–202. doi: 10.1016/0022-2836(85)90212-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Chattoraj D. K., Mason R. J., Wickner S. H. Mini-P1 plasmid replication: the autoregulation-sequestration paradox. Cell. 1988 Feb 26;52(4):551–557. doi: 10.1016/0092-8674(88)90468-0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Chattoraj D., Cordes K., Abeles A. Plasmid P1 replication: negative control by repeated DNA sequences. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6456–6460. doi: 10.1073/pnas.81.20.6456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Durland R. H., Helinski D. R. Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number. J Bacteriol. 1990 Jul;172(7):3849–3858. doi: 10.1128/jb.172.7.3849-3858.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kelly T. J. SV40 DNA replication. J Biol Chem. 1988 Dec 5;263(34):17889–17892. [PubMed] [Google Scholar]
  15. Kittell B. L., Helinski D. R. Iteron inhibition of plasmid RK2 replication in vitro: evidence for intermolecular coupling of replication origins as a mechanism for RK2 replication control. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1389–1393. doi: 10.1073/pnas.88.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lahue R. S., Su S. S., Modrich P. Requirement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1482–1486. doi: 10.1073/pnas.84.6.1482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McEachern M. J., Bott M. A., Tooker P. A., Helinski D. R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942–7946. doi: 10.1073/pnas.86.20.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  20. Pal S. K., Chattoraj D. K. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol. 1988 Aug;170(8):3554–3560. doi: 10.1128/jb.170.8.3554-3560.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Roberts J. M., Weintraub H. Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids. Cell. 1986 Aug 29;46(5):741–752. doi: 10.1016/0092-8674(86)90350-8. [DOI] [PubMed] [Google Scholar]
  22. Sternberg N., Austin S. Isolation and characterization of P1 minireplicons, lambda-P1:5R and lambda-P1:5L. J Bacteriol. 1983 Feb;153(2):800–812. doi: 10.1128/jb.153.2.800-812.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Trawick J. D., Kline B. C. A two-stage molecular model for control of mini-F replication. Plasmid. 1985 Jan;13(1):59–69. doi: 10.1016/0147-619x(85)90056-3. [DOI] [PubMed] [Google Scholar]
  24. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  25. Yates J., Warren N., Reisman D., Sugden B. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3806–3810. doi: 10.1073/pnas.81.12.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]

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