Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1986 Jul;167(1):130–137. doi: 10.1128/jb.167.1.130-137.1986

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3'----5' exonuclease (proofreading) activity.

R G Fowler, R M Schaaper, B W Glickman
PMCID: PMC212851  PMID: 3522541

Abstract

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).

Full text

PDF
130

Selected References

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

  1. Cheung S., Arndt K., Lu P. Correlation of lac operator DNA imino proton exchange kinetics with its function. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3665–3669. doi: 10.1073/pnas.81.12.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Choy H. E., Fowler R. G. The specificity of base-pair substitution induced by the mutL and mutS mutators in E. coli. Mutat Res. 1985 Mar;142(3):93–97. doi: 10.1016/0165-7992(85)90046-6. [DOI] [PubMed] [Google Scholar]
  3. Coulondre C., Miller J. H., Farabaugh P. J., Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature. 1978 Aug 24;274(5673):775–780. doi: 10.1038/274775a0. [DOI] [PubMed] [Google Scholar]
  4. Coulondre C., Miller J. H. Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues. J Mol Biol. 1977 Dec 15;117(3):525–567. doi: 10.1016/0022-2836(77)90056-0. [DOI] [PubMed] [Google Scholar]
  5. Coulondre C., Miller J. H. Genetic studies of the lac repressor. IV. Mutagenic specificity in the lacI gene of Escherichia coli. J Mol Biol. 1977 Dec 15;117(3):577–606. doi: 10.1016/0022-2836(77)90059-6. [DOI] [PubMed] [Google Scholar]
  6. Cox E. C., Horner D. L. Dominant mutators in Escherichia coli. Genetics. 1982 Jan;100(1):7–18. doi: 10.1093/genetics/100.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cox E. C., Horner D. L. Structure and coding properties of a dominant Escherichia coli mutator gene, mutD. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2295–2299. doi: 10.1073/pnas.80.8.2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Degnen G. E., Cox E. C. Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol. 1974 Feb;117(2):477–487. doi: 10.1128/jb.117.2.477-487.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DiFrancesco R., Bhatnagar S. K., Brown A., Bessman M. J. The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD. J Biol Chem. 1984 May 10;259(9):5567–5573. [PubMed] [Google Scholar]
  10. Drake J. W. Comparative rates of spontaneous mutation. Nature. 1969 Mar 22;221(5186):1132–1132. doi: 10.1038/2211132a0. [DOI] [PubMed] [Google Scholar]
  11. Echols H., Lu C., Burgers P. M. Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2189–2192. doi: 10.1073/pnas.80.8.2189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Erlich H. A., Cox E. C. Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector. Mol Gen Genet. 1980;178(3):703–708. doi: 10.1007/BF00337881. [DOI] [PubMed] [Google Scholar]
  13. Farabaugh P. J., Schmeissner U., Hofer M., Miller J. H. Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli. J Mol Biol. 1978 Dec 25;126(4):847–857. doi: 10.1016/0022-2836(78)90023-2. [DOI] [PubMed] [Google Scholar]
  14. Farabaugh P. J. Sequence of the lacI gene. Nature. 1978 Aug 24;274(5673):765–769. doi: 10.1038/274765a0. [DOI] [PubMed] [Google Scholar]
  15. Fowler R. G., Degnen G. E., Cox E. C. Mutational specificity of a conditional Escherichia coli mutator, mutD5. Mol Gen Genet. 1974;133(3):179–191. doi: 10.1007/BF00267667. [DOI] [PubMed] [Google Scholar]
  16. Gilbert W., Müller-Hill B. Isolation of the lac repressor. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1891–1898. doi: 10.1073/pnas.56.6.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glickman B. W., Radman M. Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063–1067. doi: 10.1073/pnas.77.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Glickman B. W. Spontaneous mutagenesis in Escherichia coli strains lacking 6-methyladenine residues in their DNA: an altered mutational spectrum in dam- mutants. Mutat Res. 1979 Jul;61(2):153–162. doi: 10.1016/0027-5107(79)90122-2. [DOI] [PubMed] [Google Scholar]
  19. Horiuchi T., Maki H., Maruyama M., Sekiguchi M. Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3770–3774. doi: 10.1073/pnas.78.6.3770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horiuchi T., Maki H., Sekiguchi M. A new conditional lethal mutator (dnaQ49) in Escherichia coli K12. Mol Gen Genet. 1978 Jul 25;163(3):277–283. doi: 10.1007/BF00271956. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A. Mutational specificity of depurination. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1494–1498. doi: 10.1073/pnas.81.5.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kunz B. A., Glickman B. W. The infidelity of conjugal DNA transfer in Escherichia coli. Genetics. 1983 Nov;105(3):489–500. doi: 10.1093/genetics/105.3.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lorenzetti R., Cesareni G., Cortese R. Frameshift mutations induced by an Escherichia coli strain carrying a mutator gene, mutD5. Mol Gen Genet. 1983;192(3):515–516. doi: 10.1007/BF00392200. [DOI] [PubMed] [Google Scholar]
  24. Maki H., Horiuchi T., Sekiguchi M. Structure and expression of the dnaQ mutator and the RNase H genes of Escherichia coli: overlap of the promoter regions. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7137–7141. doi: 10.1073/pnas.80.23.7137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maquat L. E., Thornton K., Reznikoff W. S. lac Promoter mutations located downstream from the transcription start site. J Mol Biol. 1980 May 25;139(3):537–549. doi: 10.1016/0022-2836(80)90145-x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Matthews B. W., Ohlendorf D. H., Anderson W. F., Takeda Y. Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1428–1432. doi: 10.1073/pnas.79.5.1428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  29. Miller J. H. Genetic studies of the lac repressor. XII. Amino acid replacements in the DNA binding domain of the Escherichia coli lac repressor. J Mol Biol. 1984 Nov 25;180(1):205–212. doi: 10.1016/0022-2836(84)90438-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Miwa J., Sadler J. R. Characterization of i-d repressor mutations of the lactose operon. J Mol Biol. 1977 Dec 25;117(4):843–868. doi: 10.1016/s0022-2836(77)80002-8. [DOI] [PubMed] [Google Scholar]
  32. Ogata R. T., Gilbert W. An amino-terminal fragment of lac repressor binds specifically to lac operator. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5851–5854. doi: 10.1073/pnas.75.12.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Radman M., Villani G., Boiteux S., Kinsella A. R., Glickman B. W., Spadari S. Replicational fidelity: mechanisms of mutation avoidance and mutation fixation. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):937–946. doi: 10.1101/sqb.1979.043.01.103. [DOI] [PubMed] [Google Scholar]
  34. Ripley L. S., Shoemaker N. B. A major role for bacteriophage T4 DNA polymerase in frameshift mutagenesis. Genetics. 1983 Mar;103(3):353–366. doi: 10.1093/genetics/103.3.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Schaaper R. M., Kunkel T. A., Loeb L. A. Infidelity of DNA synthesis associated with bypass of apurinic sites. Proc Natl Acad Sci U S A. 1983 Jan;80(2):487–491. doi: 10.1073/pnas.80.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Scheuermann R. H., Echols H. A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scheuermann R., Tam S., Burgers P. M., Lu C., Echols H. Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7085–7089. doi: 10.1073/pnas.80.23.7085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Streisinger G., Okada Y., Emrich J., Newton J., Tsugita A., Terzaghi E., Inouye M. Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol. 1966;31:77–84. doi: 10.1101/sqb.1966.031.01.014. [DOI] [PubMed] [Google Scholar]
  41. Topal M. D., DiGuiseppi S. R., Sinha N. K. Molecular basis for substitution mutations. Effect of primer terminal and template residues on nucleotide selection by phage T4 DNA polymerase in vitro. J Biol Chem. 1980 Dec 25;255(24):11717–11724. [PubMed] [Google Scholar]
  42. Topal M. D., Fresco J. R. Complementary base pairing and the origin of substitution mutations. Nature. 1976 Sep 23;263(5575):285–289. doi: 10.1038/263285a0. [DOI] [PubMed] [Google Scholar]
  43. Vaccaro K. K., Siegel E. C. The frameshift mutability of polA1 and recA1 derivatives of mutator strains of Escherichia coli. Mutat Res. 1977 Mar;42(3):443–446. doi: 10.1016/s0027-5107(77)80048-1. [DOI] [PubMed] [Google Scholar]
  44. Weber I. T., McKay D. B., Steitz T. A. Two helix DNA binding motif of CAP found in lac repressor and gal repressor. Nucleic Acids Res. 1982 Aug 25;10(16):5085–5102. doi: 10.1093/nar/10.16.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yanofsky C., Ito J., Horn V. Amino acid replacements and the genetic code. Cold Spring Harb Symp Quant Biol. 1966;31:151–162. doi: 10.1101/sqb.1966.031.01.023. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES