Abstract
We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the α subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rif(r), lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by ~3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A·T -> T·A are affected most strongly (~6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A·T -> G·C base-pair substitution hotspot in the lac operator is also reduced (~5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.
Full Text
The Full Text of this article is available as a PDF (904.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cupples C. G., Miller J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5345–5349. doi: 10.1073/pnas.86.14.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drake J. W., Allen E. F., Forsberg S. A., Preparata R. M., Greening E. O. Genetic control of mutation rates in bacteriophageT4. Nature. 1969 Mar 22;221(5186):1128–1132. [PubMed] [Google Scholar]
- Halliday J. A., Zielenska M., Awadallah S. S., Glickman B. W. Colony hybridisation in Escherichia coli: a rapid procedure for determining the distribution of specific classes of mutations among a number of preselected sites. Environ Mol Mutagen. 1990;16(3):143–148. doi: 10.1002/em.2850160303. [DOI] [PubMed] [Google Scholar]
- Kupchella E., Cebula T. A. Analysis of Salmonella typhimurium hisD3052 revertants: the use of oligodeoxyribonucleotide colony hybridization, PCR, and direct sequencing in mutational analysis. Environ Mol Mutagen. 1991;18(4):224–230. doi: 10.1002/em.2850180404. [DOI] [PubMed] [Google Scholar]
- McHenry C. S. DNA polymerase III holoenzyme. Components, structure, and mechanism of a true replicative complex. J Biol Chem. 1991 Oct 15;266(29):19127–19130. [PubMed] [Google Scholar]
- Michaels M. L., Cruz C., Grollman A. P., Miller J. H. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7022–7025. doi: 10.1073/pnas.89.15.7022. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller J. H., Low K. B. Specificity of mutagenesis resulting from the induction of the SOS system in the absence of mutagenic treatment. Cell. 1984 Jun;37(2):675–682. doi: 10.1016/0092-8674(84)90400-8. [DOI] [PubMed] [Google Scholar]
- Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
- Reha-Krantz L. J., Nonay R. L. Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases. J Biol Chem. 1994 Feb 25;269(8):5635–5643. [PubMed] [Google Scholar]
- Ripley L. S., Glickman B. W., Shoemaker N. B. Mutator versus antimutator activity of a T4 DNA polymerase mutant distinguishes two different frameshifting mechanisms. Mol Gen Genet. 1983;189(1):113–117. doi: 10.1007/BF00326062. [DOI] [PubMed] [Google Scholar]
- Ripley L. S. Transversion mutagenesis in bacteriophage T4. Mol Gen Genet. 1975 Nov 3;141(1):23–40. doi: 10.1007/BF00332376. [DOI] [PubMed] [Google Scholar]
- Schaaper R. M., Danforth B. N., Glickman B. W. Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene. J Mol Biol. 1986 May 20;189(2):273–284. doi: 10.1016/0022-2836(86)90509-7. [DOI] [PubMed] [Google Scholar]
- Schaaper R. M., Danforth B. N., Glickman B. W. Rapid repeated cloning of mutant lac repressor genes. Gene. 1985;39(2-3):181–189. doi: 10.1016/0378-1119(85)90312-9. [DOI] [PubMed] [Google Scholar]
- Schaaper R. M., Dunn R. L. Escherichia coli mutT mutator effect during in vitro DNA synthesis. Enhanced A.G replicational errors. J Biol Chem. 1987 Dec 5;262(34):16267–16270. [PubMed] [Google Scholar]
- Schaaper R. M., Dunn R. L. Spontaneous mutation in the Escherichia coli lacI gene. Genetics. 1991 Oct;129(2):317–326. doi: 10.1093/genetics/129.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spacciapoli P., Nossal N. G. A single mutation in bacteriophage T4 DNA polymerase (A737V, tsL141) decreases its processivity as a polymerase and increases its processivity as a 3'-->5' exonuclease. J Biol Chem. 1994 Jan 7;269(1):438–446. [PubMed] [Google Scholar]
- Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]