Logo of embojLink to Publisher's site
EMBO J. Oct 1992; 11(10): 3629–3634.
PMCID: PMC556822

DNA transcription and repressor binding affect deletion formation in Escherichia coli plasmids.

Abstract

Chimeric plasmids containing phage M13 and plasmid pBR322 sequences undergo deletions in Escherichia coli with a high frequency. In all plasmids one deletion endpoint is the M13 replication origin nick site. We examined the effects of transcription on the position of the other deletion end-point, by inserting in the plasmids an inducible promoter followed by a transcription terminator. Transcription dramatically affected deletions in an orientation-dependent way, such that greater than 95% of end-points were localized downstream from the inserted promoter when it faced the major plasmid transcripts. The end-points were not constrained to the transcribed region and were not affected by the orientation of pBR322 DNA replication. We propose that deletion events occur preferentially in a plasmid domain which is rendered positively supercoiled by convergent transcription. We also show that interaction of LacI repressor with the cognate operator generates a localized deletion hot spot. This hot spot is dependent on pBR322 replication, and therefore probably acts by arresting progression of DNA replication.

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 (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Lee J, Goldfarb A. lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter. Cell. 1991 Aug 23;66(4):793–798. [PubMed]
  • Liu LF, Wang JC. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. [PMC free article] [PubMed]
  • Lockshon D, Morris DR. Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization. J Mol Biol. 1985 Jan 5;181(1):63–74. [PubMed]
  • Mellon I, Hanawalt PC. Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature. 1989 Nov 2;342(6245):95–98. [PubMed]
  • Michel B, Ehrlich SD. Illegitimate recombination at the replication origin of bacteriophage M13. Proc Natl Acad Sci U S A. 1986 May;83(10):3386–3390. [PMC free article] [PubMed]
  • Michel B, D'Alençon E, Ehrlich SD. Deletion hot spots in chimeric Escherichia coli plasmids. J Bacteriol. 1989 Apr;171(4):1846–1853. [PMC free article] [PubMed]
  • Miura-Masuda A, Ikeda H. The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo. Mol Gen Genet. 1990 Feb;220(3):345–352. [PubMed]
  • Oehler S, Eismann ER, Krämer H, Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990 Apr;9(4):973–979. [PMC free article] [PubMed]
  • Pruss GJ, Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. [PubMed]
  • Rahmouni AR, Wells RD. Direct evidence for the effect of transcription on local DNA supercoiling in vivo. J Mol Biol. 1992 Jan 5;223(1):131–144. [PubMed]
  • Scholtissek S, Grosse F. A cloning cartridge of lambda t(o) terminator. Nucleic Acids Res. 1987 Apr 10;15(7):3185–3185. [PMC free article] [PubMed]
  • Skarstad K, Baker TA, Kornberg A. Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA--DNA hybrid. EMBO J. 1990 Jul;9(7):2341–2348. [PMC free article] [PubMed]
  • Smerdon MJ, Thoma F. Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell. 1990 May 18;61(4):675–684. [PubMed]
  • Straney SB, Crothers DM. Lac repressor is a transient gene-activating protein. Cell. 1987 Dec 4;51(5):699–707. [PubMed]
  • Stueber D, Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. [PMC free article] [PubMed]
  • Sutcliffe JG. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 1978 Aug;5(8):2721–2728. [PMC free article] [PubMed]
  • Thomas BJ, Rothstein R. Sex, maps, and imprinting. Cell. 1991 Jan 11;64(1):1–3. [PubMed]
  • van Wezenbeek PM, Hulsebos TJ, Schoenmakers JG. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. [PubMed]
  • Backman K, Boyer HW. Tetracycline resistance determined by pBR322 is mediated by one polypeptide. Gene. 1983 Dec;26(2-3):197–203. [PubMed]
  • Bierne H, Ehrlich SD, Michel B. The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot. EMBO J. 1991 Sep;10(9):2699–2705. [PMC free article] [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. [PMC free article] [PubMed]
  • Brown S, Ferm J, Woody S, Gussin G. Selection for mutations in the PR promoter of bacteriophage lambda. Nucleic Acids Res. 1990 Oct 25;18(20):5961–5967. [PMC free article] [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Davis BD. Transcriptional bias: a non-Lamarckian mechanism for substrate-induced mutations. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5005–5009. [PMC free article] [PubMed]
  • Gros MF, te Riele H, Ehrlich SD. Replication origin of a single-stranded DNA plasmid pC194. EMBO J. 1989 Sep;8(9):2711–2716. [PMC free article] [PubMed]
  • Hill TM, Pelletier AJ, Tecklenburg ML, Kuempel PL. Identification of the DNA sequence from the E. coli terminus region that halts replication forks. Cell. 1988 Nov 4;55(3):459–466. [PubMed]
  • Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. [PMC free article] [PubMed]
  • Horinouchi S, Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. [PMC free article] [PubMed]
  • Ikeda H, Moriya K, Matsumoto T. In vitro study of illegitimate recombination: involvement of DNA gyrase. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):399–408. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...