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. 1987 Sep;169(9):3898–3903. doi: 10.1128/jb.169.9.3898-3903.1987

Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12.

R E Braun, K O'Day, A Wright
PMCID: PMC213684  PMID: 3040665

Abstract

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Braun R. E., O'Day K., Wright A. Autoregulation of the DNA replication gene dnaA in E. coli K-12. Cell. 1985 Jan;40(1):159–169. doi: 10.1016/0092-8674(85)90319-8. [DOI] [PubMed] [Google Scholar]
  2. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chakraborty T., Yoshinaga K., Lother H., Messer W. Purification of the E. coli dnaA gene product. EMBO J. 1982;1(12):1545–1549. doi: 10.1002/j.1460-2075.1982.tb01353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chesney R. H., Scott J. R. Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7. Plasmid. 1978 Feb;1(2):145–163. doi: 10.1016/0147-619x(78)90035-5. [DOI] [PubMed] [Google Scholar]
  5. Churchward G., Holmans P., Bremer H. Increased expression of the dnaA gene has no effect on DNA replication in a dnaA+ strain of Escherichia coli. Mol Gen Genet. 1983;192(3):506–508. doi: 10.1007/BF00392197. [DOI] [PubMed] [Google Scholar]
  6. Fralick J. A. Studies on the regulation of initiation of chromosome replication in Escherichia coli. J Mol Biol. 1978 Jul 5;122(3):271–286. doi: 10.1016/0022-2836(78)90190-0. [DOI] [PubMed] [Google Scholar]
  7. Fuller R. S., Funnell B. E., 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. doi: 10.1016/0092-8674(84)90284-8. [DOI] [PubMed] [Google Scholar]
  8. Fuller R. S., 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. doi: 10.1073/pnas.80.19.5817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hansen E. B., Atlung T., Hansen F. G., Skovgaard O., von Meyenburg K. Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli. Mol Gen Genet. 1984;196(3):387–396. doi: 10.1007/BF00436184. [DOI] [PubMed] [Google Scholar]
  10. Hansen E. B., Hansen F. G., von Meyenburg K. The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12. Nucleic Acids Res. 1982 Nov 25;10(22):7373–7385. doi: 10.1093/nar/10.22.7373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hansen F. G., Rasmussen K. V. Regulation of the dnaA product in Escherichia coli. Mol Gen Genet. 1977 Oct 20;155(2):219–225. doi: 10.1007/BF00393163. [DOI] [PubMed] [Google Scholar]
  12. Hirota Y., Mordoh J., Jacob F. On the process of cellular division in Escherichia coli. 3. Thermosensitive mutants of Escherichia coli altered in the process of DNA initiation. J Mol Biol. 1970 Nov 14;53(3):369–387. doi: 10.1016/0022-2836(70)90072-0. [DOI] [PubMed] [Google Scholar]
  13. Kellenberger-Gujer G., Podhajska A. J., Caro L. A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature. Mol Gen Genet. 1978 Jun 1;162(1):9–16. doi: 10.1007/BF00333845. [DOI] [PubMed] [Google Scholar]
  14. Kingsbury D. T., Helinski D. R. DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1. Biochem Biophys Res Commun. 1970 Dec 24;41(6):1538–1544. doi: 10.1016/0006-291x(70)90562-0. [DOI] [PubMed] [Google Scholar]
  15. Kung F. C., Glaser D. A. dnaA acts before dnaC in the initiation of DNA replication. J Bacteriol. 1978 Feb;133(2):755–762. doi: 10.1128/jb.133.2.755-762.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindahl G., Hirota Y., Jacob F. On the process of cellular division in Escherichia coli: replication of the bacterial chromosome under control of prophage P2. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2407–2411. doi: 10.1073/pnas.68.10.2407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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. doi: 10.1016/0022-2836(85)90299-2. [DOI] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  19. Miki T., Kimura M., Hiraga S., Nagata T., Yura T. Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome. J Bacteriol. 1979 Dec;140(3):817–824. doi: 10.1128/jb.140.3.817-824.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nishimura A., Nishimura Y., Caro L. Isolation of Hfr strains from R+ and ColV2+ strains of Escherichia coli and derivation of an R'lac factor by transduction. J Bacteriol. 1973 Dec;116(3):1107–1112. doi: 10.1128/jb.116.3.1107-1112.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nishimura Y., Caro L., Berg C. M., Hirota Y. Chromosome replication in Escherichia coli. IV. Control of chromosome replication and cell division by an integrated episome. J Mol Biol. 1971 Feb 14;55(3):441–456. doi: 10.1016/0022-2836(71)90328-7. [DOI] [PubMed] [Google Scholar]
  22. Ohmori H., Kimura M., Nagata T., Sakakibara Y. Structural analysis of the dnaA and dnaN genes of Escherichia coli. Gene. 1984 May;28(2):159–170. doi: 10.1016/0378-1119(84)90253-1. [DOI] [PubMed] [Google Scholar]
  23. Sakakibara Y., Mizukami T. A temperature-sensitive Escherichia coli mutant defective in DNA replication: dnaN, a new gene adjacent to the dnaA gene. Mol Gen Genet. 1980;178(3):541–553. doi: 10.1007/BF00337859. [DOI] [PubMed] [Google Scholar]
  24. Sako T., Sakakibara Y. Coordinate expression of Escherichia coli dnaA and dnaN genes. Mol Gen Genet. 1980;179(3):521–526. doi: 10.1007/BF00271741. [DOI] [PubMed] [Google Scholar]
  25. Schaus N., O'Day K., Peters W., Wright A. Isolation and characterization of amber mutations in gene dnaA of escherichia coli K-12. J Bacteriol. 1981 Feb;145(2):904–913. doi: 10.1128/jb.145.2.904-913.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sotomura M., Yoshikawa M. Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K. J Bacteriol. 1975 May;122(2):623–628. doi: 10.1128/jb.122.2.623-628.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu R. Nucleotide sequence analysis of DNA. I. Partial sequence of the cohesive ends of bacteriophage lambda and 186 DNA. J Mol Biol. 1970 Aug;51(3):501–521. doi: 10.1016/0022-2836(70)90004-5. [DOI] [PubMed] [Google Scholar]
  28. Zyskind J. W., Deen L. T., Smith D. W. Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase. J Bacteriol. 1977 Mar;129(3):1466–1475. doi: 10.1128/jb.129.3.1466-1475.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

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