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EMBO J. 1989 Dec 20; 8(13): 4335–4344.
PMCID: PMC401646

The DNA unwinding element: a novel, cis-acting component that facilitates opening of the Escherichia coli replication origin.

Abstract

We have discovered that DNA supercoiling, in the absence of replication proteins, induces localized unwinding in the Escherichia coli replication origin (oriC) at the same sequence opened by the dnaA initiator protein. The DNA helix at the tandemly repeated, 13mer sequence is thermodynamically unstable, as evidenced by hypersensitivity to single-strand-specific nuclease in a negatively supercoiled plasmid, and demonstrated by stable DNA unwinding seen after two-dimensional gel electrophoresis of topoisomers. A replication-defective oriC mutant lacking the leftmost 13mer shows no nuclease hypersensitivity in two remaining 13mers and no detectable DNA unwinding on two-dimensional gels. The replication defect in the oriC mutant can be corrected by inserting a dissimilar DNA sequence with reduced helical stability in place of the leftmost 13mer. Thus, the helical instability of the leftmost 13mer, not the specific 13mer sequence, is essential for origin function. The rightmost 13mer exhibits helical instability but differs from the leftmost 13mer in its strict sequence conservation among related bacterial origins. The repeated 13mer region appears to serve two overlapping functions: protein recognition and helical instability. We propose that the cis-acting sequence whose helical instability is required for origin function be called the DNA unwinding element (DUE).

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  • Asada K, Sugimoto K, Oka A, Takanami M, Hirota Y. Structure of replication origin of the Escherichia coli K-12 chromosome: the presence of spacer sequences in the ori region carrying information for autonomous replication. Nucleic Acids Res. 1982 Jun 25;10(12):3745–3754. [PMC free article] [PubMed]
  • Baker TA, Kornberg A. Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell. 1988 Oct 7;55(1):113–123. [PubMed]
  • 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]
  • Baker TA, Funnell BE, 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]
  • Bird RE, Louarn J, Martuscelli J, Caro L. Origin and sequence of chromosome replication in Escherichia coli. J Mol Biol. 1972 Oct 14;70(3):549–566. [PubMed]
  • 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. [PubMed]
  • Borowiec JA, Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 1988 Oct;7(10):3149–3158. [PMC free article] [PubMed]
  • Brewer BJ. When polymerases collide: replication and the transcriptional organization of the E. coli chromosome. Cell. 1988 Jun 3;53(5):679–686. [PubMed]
  • Cesareni G, Muesing MA, Polisky B. Control of ColE1 DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6313–6317. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Dodson M, Echols H, Wickner S, Alfano C, Mensa-Wilmot K, Gomes B, LeBowitz J, Roberts JD, McMacken R. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7638–7642. [PMC free article] [PubMed]
  • Echols H. Multiple DNA-protein interactions governing high-precision DNA transactions. Science. 1986 Sep 5;233(4768):1050–1056. [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]
  • Fuller RS, Funnell BE, 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. [PubMed]
  • Funnell BE, Baker TA, Kornberg A. In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome. J Biol Chem. 1987 Jul 25;262(21):10327–10334. [PubMed]
  • Gellert M, O'Dea MH, Mizuuchi K. Slow cruciform transitions in palindromic DNA. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5545–5549. [PMC free article] [PubMed]
  • Hohlfeld R, Vielmetter W. Bidirectional growth of the E. coli chromosome. Nat New Biol. 1973 Apr 4;242(118):130–132. [PubMed]
  • Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. [PubMed]
  • Keller W. Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4876–4880. [PMC free article] [PubMed]
  • Kowalski D. Changes in site specificity of single-strand-specific endonucleases on supercoiled PM2 DNA with temperature and ionic environment. Nucleic Acids Res. 1984 Sep 25;12(18):7071–7086. [PMC free article] [PubMed]
  • Kowalski D, Sanford JP. Action of mung bean nuclease on supercoiled PM2 DNA. J Biol Chem. 1982 Jul 10;257(13):7820–7825. [PubMed]
  • Kowalski D, Kroeker WD, Laskowski M., Sr Mung bean nuclease I. Physical, chemical, and catalytic properties. Biochemistry. 1976 Oct 5;15(20):4457–4463. [PubMed]
  • Kowalski D, Natale DA, Eddy MJ. Stable DNA unwinding, not "breathing," accounts for single-strand-specific nuclease hypersensitivity of specific A+T-rich sequences. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9464–9468. [PMC free article] [PubMed]
  • Lark KG. Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15T. J Mol Biol. 1972 Feb 28;64(1):47–60. [PubMed]
  • LeBowitz JH, McMacken R. The Escherichia coli dnaB replication protein is a DNA helicase. J Biol Chem. 1986 Apr 5;261(10):4738–4748. [PubMed]
  • Leonard AC, Helmstetter CE. Cell cycle-specific replication of Escherichia coli minichromosomes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5101–5105. [PMC free article] [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]
  • Louarn J, Bouché JP, Patte J, Louarn JM. Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli. Mol Gen Genet. 1984;195(1-2):170–174. [PubMed]
  • Matsui M, Oka A, Takanami M, Yasuda S, Hirota Y. Sites of dnaA protein-binding in the replication origin of the Escherichia coli K-12 chromosome. J Mol Biol. 1985 Aug 5;184(3):529–533. [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Meijer M, Beck E, Hansen FG, Bergmans HE, Messer W, von Meyenburg K, Schaller H. Nucleotide sequence of the origin of replication of the Escherichia coli K-12 chromosome. Proc Natl Acad Sci U S A. 1979 Feb;76(2):580–584. [PMC free article] [PubMed]
  • Messer W, Bergmans HE, Meijer M, Womack JE, Hansen FG, von Meyenburg K. Mini-chromosomes: plasmids which carry the E. coli replication origin. Mol Gen Genet. 1978 Jul 4;162(3):269–275. [PubMed]
  • Miki T, Hiraga S, Nagata T, Yura T. Bacteriophage lambda carrying the Escherichia coli chromosomal region of the replication origin. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5099–5103. [PMC free article] [PubMed]
  • Oka A, Sugimoto K, Takanami M, Hirota Y. Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol Gen Genet. 1980 Apr;178(1):9–20. [PubMed]
  • Oka A, Sasaki H, Sugimoto K, Takanami M. Sequence organization of replication origin of the Escherichia coli K-12 chromosome. J Mol Biol. 1984 Jul 15;176(4):443–458. [PubMed]
  • Pruss GJ, Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. [PubMed]
  • Radloff R, Bauer W, Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. [PMC free article] [PubMed]
  • Schnos M, Zahn K, Inman RB, Blattner FR. Initiation protein induced helix destabilization at the lambda origin: a prepriming step in DNA replication. Cell. 1988 Feb 12;52(3):385–395. [PubMed]
  • Sheflin LG, Kowalski D. Mung bean nuclease cleavage of a dA + dT-rich sequence or an inverted repeat sequence in supercoiled PM2 DNA depends on ionic environment. Nucleic Acids Res. 1984 Sep 25;12(18):7087–7104. [PMC free article] [PubMed]
  • Sheflin LG, Kowalski D. Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA. Nucleic Acids Res. 1985 Sep 11;13(17):6137–6154. [PMC free article] [PubMed]
  • Sugimoto K, Oka A, Sugisaki H, Takanami M, Nishimura A, Yasuda Y, Hirota Y. Nucleotide sequence of Escherichia coli K-12 replication origin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):575–579. [PMC free article] [PubMed]
  • Umek RM, Kowalski D. Yeast regulatory sequences preferentially adopt a non-B conformation in supercoiled DNA. Nucleic Acids Res. 1987 Jun 11;15(11):4467–4480. [PMC free article] [PubMed]
  • Umek RM, Kowalski D. The ease of DNA unwinding as a determinant of initiation at yeast replication origins. Cell. 1988 Feb 26;52(4):559–567. [PubMed]
  • van der Ende A, Baker TA, Ogawa T, Kornberg A. Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3954–3958. [PMC free article] [PubMed]
  • von Meyenburg K, Hansen FG, Nielsin LD, Riise E. Origin of replication, oriC, or the Escherichia coli chromosome on specialized transducing phages lambda asn. Mol Gen Genet. 1978 Apr 17;160(3):287–295. [PubMed]
  • Wang JC, Peck LJ, Becherer K. DNA supercoiling and its effects on DNA structure and function. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 1):85–91. [PubMed]
  • Wu HY, Shyy SH, Wang JC, Liu LF. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. [PubMed]
  • Yasuda S, Hirota Y. Cloning and mapping of the replication origin of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5458–5462. [PMC free article] [PubMed]
  • Yung BY, 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]
  • 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]

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