• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Oct 1987; 169(10): 4703–4709.
PMCID: PMC213843

DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli.


The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.7M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baker TA, Sekimizu K, Funnell BE, Kornberg A. Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell. 1986 Apr 11;45(1):53–64. [PubMed]
  • Bolivar F, Rodriguez RL, Betlach MC, Boyer HW. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Burkardt HJ, Riess G, Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. [PubMed]
  • Cohen SN, Chang AC. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734–737. [PMC free article] [PubMed]
  • Cross MA, Warne SR, Thomas CM. Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis. Plasmid. 1986 Mar;15(2):132–146. [PubMed]
  • Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. [PMC free article] [PubMed]
  • Figurski DH, Pohlman RF, Bechhofer DH, Prince AS, Kelton CA. Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1935–1939. [PMC free article] [PubMed]
  • Figurski DH, Young C, Schreiner HC, Pohlman RF, Bechhofer DH, Prince AS, D'Amico TF. Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon. Basic Life Sci. 1985;30:227–241. [PubMed]
  • Frey J, Chandler M, Caro L. The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC. Mol Gen Genet. 1979 Jul 13;174(2):117–126. [PubMed]
  • Fuller RS, 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Germino J, Bastia D. Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4692–4696. [PMC free article] [PubMed]
  • Guiney DG, Chikami G, Deiss C, Yakobson E. The origin of plasmid DNA transfer during bacterial conjugation. Basic Life Sci. 1985;30:521–534. [PubMed]
  • Hansen EB, Yarmolinsky MB. 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. [PMC free article] [PubMed]
  • Junker DE, Jr, Rokeach LA, Ganea D, Chiaramello A, Zyskind JW. Transcription termination within the Escherichia coli origin of DNA replication, oriC. Mol Gen Genet. 1986 Apr;203(1):101–109. [PubMed]
  • Kline BC, Kogoma T, Tam JE, Shields MS. Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance. J Bacteriol. 1986 Oct;168(1):440–443. [PMC free article] [PubMed]
  • Lanka E, Lurz R, Fürste JP. Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid. 1983 Nov;10(3):303–307. [PubMed]
  • McKay RD. Binding of a simian virus 40 T antigen-related protein to DNA. J Mol Biol. 1981 Jan 25;145(3):471–488. [PubMed]
  • Monk M, Kinross J. Conditional lethality of recA and recB derivatives of a strain of Escherichia coli K-12 with a temperature-sensitive deoxyribonucleic acid polymerase I. J Bacteriol. 1972 Mar;109(3):971–978. [PMC free article] [PubMed]
  • Moskaluk C, Bastia D. The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1215–1218. [PMC free article] [PubMed]
  • Murakami Y, Ohmori H, Yura T, Nagata T. Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication. J Bacteriol. 1987 Apr;169(4):1724–1730. [PMC free article] [PubMed]
  • Pohlman RF, Figurski DH. Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA. J Bacteriol. 1983 Nov;156(2):584–591. [PMC free article] [PubMed]
  • Schmidhauser TJ, Helinski DR. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. [PMC free article] [PubMed]
  • Schreiner HC, Bechhofer DH, Pohlman RF, Young C, Borden PA, Figurski DH. Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA. J Bacteriol. 1985 Jul;163(1):228–237. [PMC free article] [PubMed]
  • Seufert W, Messer W. DnaA protein binding to the plasmid origin region can substitute for primosome assembly during replication of pBR322 in vitro. Cell. 1987 Jan 16;48(1):73–78. [PubMed]
  • Smith CA, Thomas CM. Molecular gentic analysis of the trfB and korB region of broad host range plasmid RK2. J Gen Microbiol. 1984 Jul;130(7):1651–1663. [PubMed]
  • Smith CA, Thomas CM. Nucleotide sequence of the trfA gene of broad host-range plasmid RK2. J Mol Biol. 1984 May 25;175(3):251–262. [PubMed]
  • Stalker DM, Thomas CM, Helinski DR. 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. [PubMed]
  • Thomas CM, Meyer R, Helinski DR. Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol. 1980 Jan;141(1):213–222. [PMC free article] [PubMed]
  • Thomas CM, Stalker DM, Helinski DR. Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):1–7. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Wu HM, Crothers DM. The locus of sequence-directed and protein-induced DNA bending. Nature. 1984 Apr 5;308(5959):509–513. [PubMed]
  • 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. [PubMed]
  • Zyskind JW, Cleary JM, Brusilow WS, Harding NE, Smith DW. 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. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...