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. 1992 Feb 25;20(4):811–817. doi: 10.1093/nar/20.4.811

Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K.

F Wu 1, I Goldberg 1, M Filutowicz 1
PMCID: PMC312022  PMID: 1627205

Abstract

A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.

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Selected References

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

  1. Atlung T., Clausen E. S., Hansen F. G. Autoregulation of the dnaA gene of Escherichia coli K12. Mol Gen Genet. 1985;200(3):442–450. doi: 10.1007/BF00425729. [DOI] [PubMed] [Google Scholar]
  2. Atlung T., Clausen E., Hansen F. G. Autorepression of the dnaA gene of Escherichia coli. Adv Exp Med Biol. 1984;179:199–207. doi: 10.1007/978-1-4684-8730-5_20. [DOI] [PubMed] [Google Scholar]
  3. Baker T. A., Funnell B. E., Kornberg A. Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro. J Biol Chem. 1987 May 15;262(14):6877–6885. [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bramhill D., Kornberg A. A model for initiation at origins of DNA replication. Cell. 1988 Sep 23;54(7):915–918. doi: 10.1016/0092-8674(88)90102-x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Chesney R. H., Scott J. R., Vapnek D. Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli. J Mol Biol. 1979 May 15;130(2):161–173. doi: 10.1016/0022-2836(79)90424-8. [DOI] [PubMed] [Google Scholar]
  11. Crosa J. H., Luttropp L. K., Falkow S. Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli. J Bacteriol. 1976 Apr;126(1):454–466. doi: 10.1128/jb.126.1.454-466.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crosa J. H. Three origins of replication are active in vivo in the R plasmid RSF1040. J Biol Chem. 1980 Dec 10;255(23):11075–11077. [PubMed] [Google Scholar]
  13. De Lucia P., Cairns J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature. 1969 Dec 20;224(5225):1164–1166. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
  14. Dellis S., Filutowicz M. Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein. J Bacteriol. 1991 Feb;173(3):1279–1286. doi: 10.1128/jb.173.3.1279-1286.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Filutowicz M., Appelt K. The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication. Nucleic Acids Res. 1988 May 11;16(9):3829–3843. doi: 10.1093/nar/16.9.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Filutowicz M., Davis G., Greener A., Helinski D. R. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. doi: 10.1093/nar/13.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Filutowicz M., Inman R. A compact nucleoprotein structure is produced by binding of Escherichia coli integration host factor (IHF) to the replication origin of plasmid R6K. J Biol Chem. 1991 Dec 15;266(35):24077–24083. [PubMed] [Google Scholar]
  18. Filutowicz M., McEachern M. J., Helinski D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9645–9649. doi: 10.1073/pnas.83.24.9645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Filutowicz M., Uhlenhopp E., Helinski D. R. Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. J Mol Biol. 1986 Jan 20;187(2):225–239. doi: 10.1016/0022-2836(86)90230-5. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Fuller R. S., Kornberg A. Purified dnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5817–5821. doi: 10.1073/pnas.80.19.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Greener A., Filutowicz M. S., McEachern M. J., Helinski D. R. N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication. Mol Gen Genet. 1990 Oct;224(1):24–32. doi: 10.1007/BF00259447. [DOI] [PubMed] [Google Scholar]
  24. Hansen E. B., Yarmolinsky M. B. Host participation in plasmid maintenance: dependence upon dnaA of replicons derived from P1 and F. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4423–4427. doi: 10.1073/pnas.83.12.4423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Inuzuka M., Wada Y. A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K. EMBO J. 1985 Sep;4(9):2301–2307. doi: 10.1002/j.1460-2075.1985.tb03930.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Kline B. C. Aspects of plasmid F maintenance in Escherichia coli. Can J Microbiol. 1988 Apr;34(4):526–535. doi: 10.1139/m88-090. [DOI] [PubMed] [Google Scholar]
  29. Kline B. C., Kogoma T., Tam J. E., Shields M. S. Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance. J Bacteriol. 1986 Oct;168(1):440–443. doi: 10.1128/jb.168.1.440-443.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kolter R., Helinski D. R. Activity of the replication terminus of plasmid R6K in hybrid replicons in Escherichia coli. J Mol Biol. 1978 Sep 25;124(3):425–441. doi: 10.1016/0022-2836(78)90180-8. [DOI] [PubMed] [Google Scholar]
  31. Kolter R., Helinski D. R. Construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid. 1978 Sep;1(4):571–580. doi: 10.1016/0147-619x(78)90014-8. [DOI] [PubMed] [Google Scholar]
  32. Kolter R., Helinski D. R. Plasmid R6K DNA replication. II. Direct nucleotide sequence repeats are required for an active gamma-origin. J Mol Biol. 1982 Oct 15;161(1):45–56. doi: 10.1016/0022-2836(82)90277-7. [DOI] [PubMed] [Google Scholar]
  33. Kontomichalou P., Mitani M., Clowes R. C. Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under either relaxed or stringent control. J Bacteriol. 1970 Oct;104(1):34–44. doi: 10.1128/jb.104.1.34-44.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lother H., Kölling R., Kücherer C., Schauzu M. dnaA protein-regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes. EMBO J. 1985 Feb;4(2):555–560. doi: 10.1002/j.1460-2075.1985.tb03664.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. MacAllister T. W., Kelley W. L., Miron A., Stenzel T. T., Bastia D. Replication of plasmid R6K origin gamma in vitro. Dependence on dual initiator proteins and inhibition by transcription. J Biol Chem. 1991 Aug 25;266(24):16056–16062. [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Mukherjee S., Erickson H., Bastia D. Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein. Cell. 1988 Feb 12;52(3):375–383. doi: 10.1016/s0092-8674(88)80030-8. [DOI] [PubMed] [Google Scholar]
  40. Mukhopadhyay P., Filutowicz M., Helinski D. R. Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins. J Biol Chem. 1986 Jul 15;261(20):9534–9539. [PubMed] [Google Scholar]
  41. Nishimura Y., Caro L., Berg C. M., Hirota Y. Chromosome replication in Escherichia coli. IV. Control of chromosome replication and cell division by an integrated episome. J Mol Biol. 1971 Feb 14;55(3):441–456. doi: 10.1016/0022-2836(71)90328-7. [DOI] [PubMed] [Google Scholar]
  42. Polaczek P., Wright A. Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box. New Biol. 1990 Jun;2(6):574–582. [PubMed] [Google Scholar]
  43. Shafferman A., Flashner Y., Hertman I., Lion M. Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins. Mol Gen Genet. 1987 Jun;208(1-2):263–270. doi: 10.1007/BF00330452. [DOI] [PubMed] [Google Scholar]
  44. Shafferman A., Helinski D. R. Structural properties of the beta origin of replication of plasmid R6K. J Biol Chem. 1983 Apr 10;258(7):4083–4090. [PubMed] [Google Scholar]
  45. 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]
  46. Shon M., Germino J., Bastia D. The nucleotide sequence of the replication origin beta of the plasmid R6K. J Biol Chem. 1982 Nov 25;257(22):13823–13827. [PubMed] [Google Scholar]
  47. Stenzel T. T., MacAllister T., Bastia D. Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101. Genes Dev. 1991 Aug;5(8):1453–1463. doi: 10.1101/gad.5.8.1453. [DOI] [PubMed] [Google Scholar]
  48. Tomizawa J., Selzer G. Initiation of DNA synthesis in Escherichia coli. Annu Rev Biochem. 1979;48:999–1034. doi: 10.1146/annurev.bi.48.070179.005031. [DOI] [PubMed] [Google Scholar]
  49. Wickner S., Hoskins J., Chattoraj D., McKenney K. Deletion analysis of the mini-P1 plasmid origin of replication and the role of Escherichia coli DnaA protein. J Biol Chem. 1990 Jul 15;265(20):11622–11627. [PubMed] [Google Scholar]
  50. Yin J. C., Reznikoff W. S. dnaA, an essential host gene, and Tn5 transposition. J Bacteriol. 1987 Oct;169(10):4637–4645. doi: 10.1128/jb.169.10.4637-4645.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yung B. Y., Kornberg A. The dnaA initiator protein binds separate domains in the replication origin of Escherichia coli. J Biol Chem. 1989 Apr 15;264(11):6146–6150. [PubMed] [Google Scholar]
  52. Zyskind J. W., Cleary J. M., Brusilow W. S., Harding N. E., Smith D. W. Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1164–1168. doi: 10.1073/pnas.80.5.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]

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