Summary
Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Atlung T, Lobner-Olsen A, Hansen FG (1987) Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli. Mol Gen Genet 206:51–59
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:513–1523
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254
Braun EB, Wright A (1986) DNA methylation differentially enhances the expression of one of the two E. coli dnaA promotors in vivo and in vitro. Mol Gen Genet 202:256–250
Buhk H-J, Messer W (1983) The replication origin region of Escherichia coli: nucleotide sequence and functional units. Gene 24:265–279
Colman A, Byers MJ, Primrose SB, Lyons A (1978) Rapid purification of plasmid DNAs by hydroxyapatite chromatography. Eur J Biochem 91:303–310
Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:7370–7374
Hansen FG, Koefoed S, von Meyenburg K, Atlung T (1981) Transcription and translation events in the oriC region of the E. coli chromosome. Symp Mol Cell Biol ICN-UCLA 22:37–55
Hansen FG, Hansen EB, Atlung T (1982) The nucleotide sequence of the dnaA gene promoter and of the adjacent rpm H gene coding for the ribosomal protein L34, of Escherichia coli. EMBO J 1:1043–1048
Howe JG, Hershey JWB (1981) A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli. J Biol Chem 256:12836–12839
Hughes P, Squali-Houssaini F, Forterre P, Kohiyama M (1984) In vitro replication of a dam methylated and nonmethylated oriC plasmid. J Mol Biol 176:155–159
Hughes P, Landoulsi A, Kohiyama M (1988) A novel role for cAMP in the control of the E. coli DNA replication initiator protein DnaA. Cell 55:343–350
Jacq A, Kern R, Tsugita A, Kohiyama M (1989) Purification and characterisation of a low molecular weight membrane protein with affinity for the Escherichia coli origin of replication. J Bacteriol 171
Kessler WS (1981) Use of protein A bearing Staphylococci for the immunoprecipitation and isolation of antigens from cells. Methods Enzymol 73:442–459
Kogoma T, von Meyenburg K (1983) The origin of replication, oriC, and the DnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K12. EMBO J 2:463–468
Laengle-Rouault F, Maenhaut-Michel G, Radman M (1986) GATC sequence and mismatch repair in Escherichia coli. EMBO J 5:2009–2013
Lin-Chao S, Bremer H (1986) Effect of bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli. Mol Gen Genet 203:143–149
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Marinus MG (1987) DNA methylation in Escherichia coli. Annu Rev Genet 21:113–131
Marinus MG, Morris NR (1974) Biological function for 6-methyladenine residues in the DNA of Escherichia coli K12. J Mol Biol 85:309–322
Marinus MG, Carraway M, Frey AZ, Brown L, Arraj JA (1983) Insertion mutation in the dam gene of Escherichia coli K12. Mol Gen Genet 191:288–289
Meijer M, Beck E, Hansen FG, Birgams HEN, Messer W, von Meyenburg K, Schaller H (1979) Nucleotide sequence of the origin of replication of Escherichia coli K-12 chromosome. Proc Natl Acad Sci USA 76:580–584
Messer W, Heimann B, Meijer M, Hall S (1980) Structure of the E. coli replication origin oriC sequence required for maintenance, establishment and bidirectional replication. Symp Mol Cell Biol ICN-UCLA 19:161–169
Messer W, Bellekes U, Lother H (1985) Effect of dam methylation on the activity of the E. coli replication origin, oriC. EMBO J 4:1327–1332
Ogden GB, Prat MJ, Schaechter M (1988) The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated. Cell 54:127–135
Ohmori H, Kimura M, Nagata T, Sakakibara Y (1984) Structural analysis of the DnaA and DnaN genes of Escherichia coli. Gene 28:159–170
Oka A, Sugimoto S, Takanami M (1980) Replication origin of the Escherichia coli chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol Gen Genet 178:9–20
Peterson KR, Westman KF, Mount D, Marinus MG (1985) Viability of Escherichia coli K12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon. Mol Gen Genet 201:14–19
Russell DW, Zinder ND (1987) Hemimethylation prevents DNA replication in E. coli. Cell 50:1071–1079
Seiler A, Blöcker H, Franck R, Kahmann R (1986) The mom gene of bacteriophage Mu: the mechanism of methylation-dependant expression. EMBO J 5:2719–2728
Smith DW, Garland AM, Herman G, Enns RE, Baker TA, Zyskind JW (1985) Importance of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli. EMBO J 4:1319–1326
Sugimoto K, Oka A, Sugisaki M, Takanami M, Nishimura A, Yasuda Y, Hirota Y (1979) Nucleotide sequence of Escherichia coli K-12 replication origin. Proc Natl Acad Sci USA 76:575–579
Sutcliffe JG (1978) Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp Quant Biol 43:77–90
Urieli-Shoval S, Gruenbaum Y, Razin A (1983) Sequence and Substrate specifity of isolated DNA methylases from Escherichia coli C. J Bacteriol 153:274–280
Wake CT, Wilson G (1981) Defined oligomeric SV40 DNA: a sensitive probe of general recombination in somatic cell. Cell 21:141–148
Yasuda S, Hirota Y (1977) Cloning and mapping of the replication origin of Escherichia coli. Proc Natl Acad Sci USA 74:5458–5462
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Landoulsi, A., Hughes, P., Kern, R. et al. dam methylation and the initiation of DNA replication on oriC plasmids. Mol Gen Genet 216, 217–223 (1989). https://doi.org/10.1007/BF00334359
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00334359