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Mutations in the DnaA binding sites of the replication origin ofEscherichia coli

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Summary

Mutations (base changes) were introduced into the four DnaA binding sites (DnaA boxes) of theEscherichia coli replication origin,oriC. Mutations in a single DnaA box did not impair the ability of these origins to replicate in vivo and in vitro. A combination of mutations in two DnaA boxes, R1 and R4, resulted in slower growth of theoriC plasmid-bearing host cells. DnaA protein interaction with mutant and wild-type DnaA boxes was analyzed by DNase I footprinting. Binding of DnaA protein to a mutated DnaA box R1 was not affected by a mutation in DnaA box R4 and vice versa. Mutations in DnaA boxes R1 and R4 did not modify the ability of the DnaA protein to bind to other DnaA boxes inoriC.

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References

  • Asai T, Takanami M, Imai M (1990) The AT richness andgid transcription determine the left border of the replication origin of theE. coli chromosome. EMBO J 9:4065–4072

    Google Scholar 

  • Atlung T, Clausen E, Hansen FG (1985) Autoregulation of thednaA gene ofEscherichia coli. Mol Gen Genet 200:442–450

    Google Scholar 

  • Bramhill D, Kornberg A (1988) Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of theE. coli chromosome. Cell 52:743–755

    Google Scholar 

  • Braun RE, O'Day K, Wright A (1985) Autoregulation of the DNA replication genednaA inE. coli. Cell 40:159–169

    Google Scholar 

  • Fukuoka T, Moriya S, Yoshikawa H, Ogasawara N (1990) Purification and characterization of an initiation protein for chromosomal replication, DnaA, inBacillus subtilis. J Biochem (Tokyo) 107:732–739

    Google Scholar 

  • Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of theE. coli chromosome. Proc Natl Acad Sci USA 78:7370–7374

    Google Scholar 

  • Fuller RS, Funnell BE, Kornberg A (1984) ThednaA protein complex with theE. coli chromosomal origin(oriC) and other sites. Cell 38:889–900

    Google Scholar 

  • Galas D, Schmitz A (1978) Dnase footprinting: A simple method for determining protein-DNA binding specificity. Nucleic Acids Res 5:3157–3170

    Google Scholar 

  • Georgopoulos C (1989) TheE. coli dnaA initiation protein: a protein for all seasons. Trends Genet 5:319–321

    Google Scholar 

  • Gielow A, Kücherer C, Kölling R, Messer W (1988) Transcription in the region of the replication origin,oriC, ofEscherichia coli. Termination ofasnC transcripts. Mol Gen Genet 214:474–481

    Google Scholar 

  • Gielow A, Diederich L, Messer W (1991) Characterization of a phage-plasmid hybrid (Phasyl) with two independent origins of replication isolated fromEscherichia coli. J Bacteriol 173:73–79

    Google Scholar 

  • Gille H, Messer W (1991) Localized unwinding and structural perturbations in the origin of replication,oriC, ofEscherichia coli in vitro andin vivo. EMBO J 10:1579–1584

    Google Scholar 

  • Gille H, Egan JB, Roth A, Messer W (1991) The FIS protein binds and bends the origin of chromosomal DNA replication,oriC, ofEscherichia coli. Nucleic Acids Res 19:4167–4172

    Google Scholar 

  • Jensen MR, Loebner-Olesen A, Rasmussen KV (1990)Escherichia coli minichromosomes: Absence of copy number control, and random segregation. J Mol Biol 215:257–265

    Google Scholar 

  • Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492

    Google Scholar 

  • Kücherer C, Lother H, Kölling R, Schauzu MA, Messer W (1986) Regulation of transcription of the chromosomaldnaA gene ofEscherichia coli. Mol Gen Genet 205:115–121

    Google Scholar 

  • Loebner-Olesen A, Atlung T, Rasmussen KV (1987) Stability and replication control ofE. coli minichromosomes. J Bacteriol 169:2835–2842

    Google Scholar 

  • Lother H, Kölling R, Kücherer C, Schauzu MA (1985) Initiation of replication inEscherichia coli involvesdnaA protein regulated transcription within the replication origin. EMBO J 4:555–560

    Google Scholar 

  • Meijer M, Beck E, Hansen FG, Bergmans HE, Messer W, von Meyenburg K, Schaller H (1979) Nucleotide sequence of the origin of replication of theE. coli K-12 chromosome. Proc Natl Acad Sci USA 76:580–584

    Google Scholar 

  • Messer W, Heimann B, Meijer M, Hall S (1980) Structure of theE. coli replication origin.oriC sequences required for maintenance, establishment and bidirectional replication. ICN-UCLA Symp Mol Cell Biol 19:161–169

    Google Scholar 

  • Messer W, Egan JB, Gille H, Holz A, Schaefer C, Woelker B (1991) The complex oforiC DNA with the DnaA initiator protein. Res Microbiol 142:119–125

    Google Scholar 

  • Messing J (1983) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Methods Enzymol 101:20–78

    Google Scholar 

  • Nozaki N, Okazaki T, Ogawa T (1988)In vitro transcription of the origin region of replication of theEscherichia coli chromosome. J Biol Chem 263:14176–14183

    Google Scholar 

  • Oka A, Sasaki H, Sugimoto K, Takanami M (1984) Sequence organization of replication origin of theEscherichia coli K-12 chromosome. J Mol Biol 176:443–458

    Google Scholar 

  • Polaczek P, Wright A (1990) Regulation of expression of thednaA gene inEscherichia coli: Role of the two promoters and the DnaA box. New Biologist 2:574–582

    Google Scholar 

  • Quinones A, Jueterbock W-R, Messer W (1991) Expression of thednaA gene ofEscherichia coli is inducible by DNA damage. Mol Gen Genet 227:9–16

    Google Scholar 

  • Rokeach LA, Chiaramello AE, Junker DE, Crain K, Nourani A, Jannatipour M, Zyskind JW (1986) Effects of DnaA protein on replication and transcription events at theEscherichia coli origin of replication,oriC. ICN-UCLA Symp Mol Cell Biol 47:415–427

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Samitt CE, Hansen FG, Miller JF, Schaechter M (1989)In vivo studies of DnaA binding to the origin of replication ofEscherichia coli. EMBO J 8:989–993

    Google Scholar 

  • Sanger F, Nicklen S, Coulse R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Schaefer C, Messer W (1988) Termination of theE. coli asnC transcript. The DnaA protein/dnaA box complex blocks transcribing RNA polymerase. Gene 73:347–354

    Google Scholar 

  • Schaefer C, Messer W (1989) Directionality of DnaA protein/DNA interaction. Active orientation of the DnaA protein/dnaA box complex in transcription termination. EMBO J 8:1609–1613

    Google Scholar 

  • Schaefer C, Messer W (1991) DnaA protein/DNA interaction. Modulation of the recognition sequence. Mol Gen Genet 226:34–40

    Google Scholar 

  • Schaefer C, Holz A, Messer W (1991) DnaA protein mediated transcription termination in thegua operon ofEscherichia coli. In: Fanning E, Hughes P, Kohiyama M (eds) Regulatory Mechanisms of DNA Replication. Springer, Berlin New York, in press

    Google Scholar 

  • Schauzu MA, Kücherer C, Kölling R, Messer W, Lother H (1987) Transcripts within the replication origin,oriC, ofEscherichia coli. Nucleic Acids Res 15:2479–2497

    Google Scholar 

  • Sekimizu K, Bramhill D, Kornberg A (1988) Sequential early stages in thein vitro initiation of replication at the origin of theEscherichia coli chromosome. J Biol Chem 263:7124–7130

    Google Scholar 

  • Seufert W, Messer W (1986) Initiation ofEscherichia coli minichromosome replication atoriC and at protein n′ recognition sites. Two modes for initiating DNA synthesisin vitro. EMBO J 5:3401–3406

    Google Scholar 

  • Stuitje AR, de Wind N, van der Spek JC, Pors TH, Meijer M (1986) Dissection of promoter sequences involved in transcriptional activation of theEscherichia coli replication origin. Nucleic Acids Res 14:2333–2344

    Google Scholar 

  • Sugimoto K, Oka A, Sugisaki H, Takanami M, Nishimura A, Yasuda Y, Hirota Y (1979) Nucleotide sequence ofEscherichia coli replication origin. Proc Natl Acad Sci USA 76:575–579

    Google Scholar 

  • Wang Q, Kaguni JM (1987) Transcriptional repression of thednaA gene ofEscherichia coli by DnaA protein. Mol Gen Genet 209:518–525

    Google Scholar 

  • Wang Q, Kaguni JM (1989) DnaA protein regulates transcription of therpoH gene ofEscherichia coli. J Biol Chem 264:7338–7344

    Google Scholar 

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Communicated by R. Devoret

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Holz, A., Schaefer, C., Gille, H. et al. Mutations in the DnaA binding sites of the replication origin ofEscherichia coli . Molec. Gen. Genet. 233, 81–88 (1992). https://doi.org/10.1007/BF00587564

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  • DOI: https://doi.org/10.1007/BF00587564

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