Abstract
DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.
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- Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bryan S. K., Moses R. E. Map location of the pcbA mutation and physiology of the mutant. J Bacteriol. 1984 Apr;158(1):216–221. doi: 10.1128/jb.158.1.216-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1038/2241164a0. [DOI] [PubMed] [Google Scholar]
- Hagensee M. E., Timme T. L., Bryan S. K., Moses R. E. DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4195–4199. doi: 10.1073/pnas.84.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Konrad E. B. Isolation of an Escherichia coli K-12 dnaE mutation as a mutator. J Bacteriol. 1978 Mar;133(3):1197–1202. doi: 10.1128/jb.133.3.1197-1202.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maki H., Horiuchi T., Sekiguchi M. Isolation of conditional lethal mutator mutants of Escherichia coli by localized mutagenesis. J Bacteriol. 1983 Mar;153(3):1361–1367. doi: 10.1128/jb.153.3.1361-1367.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maruyama M., Horiuchi T., Maki H., Sekiguchi M. A dominant (mutD5) and a recessive (dnaQ49) mutator of Escherichia coli. J Mol Biol. 1983 Jul 15;167(4):757–771. doi: 10.1016/s0022-2836(83)80109-0. [DOI] [PubMed] [Google Scholar]
- McHenry C. S. DNA polymerase III holoenzyme of Escherichia coli. Annu Rev Biochem. 1988;57:519–550. doi: 10.1146/annurev.bi.57.070188.002511. [DOI] [PubMed] [Google Scholar]
- McHenry C., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Biol Chem. 1977 Sep 25;252(18):6478–6484. [PubMed] [Google Scholar]
- Morrison D. A. Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol. 1977 Oct;132(1):349–351. doi: 10.1128/jb.132.1.349-351.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niwa O., Bryan S. K., Moses R. E. Alternate pathways of DNA replication: DNA polymerase I-dependent replication. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7024–7027. doi: 10.1073/pnas.78.11.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niwa O., Bryan S. K., Moses R. E. Replication at restrictive temperatures in Escherichia coli containing a polCts mutation. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5572–5576. doi: 10.1073/pnas.76.11.5572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schekman R., Weiner A., Kornberg A. Multienzyme systems of DNA replication. Science. 1974 Dec 13;186(4168):987–993. doi: 10.1126/science.186.4168.987. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]