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. 1992 Oct 11;20(19):5119–5125. doi: 10.1093/nar/20.19.5119

A comparison of the fidelity of copying 5-methylcytosine and cytosine at a defined DNA template site.

J C Shen 1, S Creighton 1, P A Jones 1, M F Goodman 1
PMCID: PMC334293  PMID: 1383939

Abstract

5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C-->T transitions, resulting from spontaneous deamination of mC to T. The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesis via G-->A transitions. Stimulation of the G-->A mutational pathway could involve preferential misincorporation of dAMP opposite template mC compared to C. To investigate this possibility, we synthesized a sequence containing mC at a defined template location. We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity. Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase. AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite mC than C. Favored extension of a 5-MeC.A over C.A mispair was also observed with a difference of about 3-fold. In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either mC or C sites or when extending mispairs involving mC and C. Incorporation of dAMP opposite either C or mC was barely detectable using pol alpha, although pol alpha has been observed to form A.C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-->A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.

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Selected References

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

  1. Boosalis M. S., Petruska J., Goodman M. F. DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem. 1987 Oct 25;262(30):14689–14696. [PubMed] [Google Scholar]
  2. Brown T. C., Jiricny J. A specific mismatch repair event protects mammalian cells from loss of 5-methylcytosine. Cell. 1987 Sep 11;50(6):945–950. doi: 10.1016/0092-8674(87)90521-6. [DOI] [PubMed] [Google Scholar]
  3. Brown T. C., Jiricny J. Different base/base mispairs are corrected with different efficiencies and specificities in monkey kidney cells. Cell. 1988 Aug 26;54(5):705–711. doi: 10.1016/s0092-8674(88)80015-1. [DOI] [PubMed] [Google Scholar]
  4. Clayton L. K., Goodman M. F., Branscomb E. W., Galas D. J. Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. J Biol Chem. 1979 Mar 25;254(6):1902–1912. [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Creighton S., Huang M. M., Cai H., Arnheim N., Goodman M. F. Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini. J Biol Chem. 1992 Feb 5;267(4):2633–2639. [PubMed] [Google Scholar]
  8. Duncan B. K., Miller J. H. Mutagenic deamination of cytosine residues in DNA. Nature. 1980 Oct 9;287(5782):560–561. doi: 10.1038/287560a0. [DOI] [PubMed] [Google Scholar]
  9. Ehrlich M., Norris K. F., Wang R. Y., Kuo K. C., Gehrke C. W. DNA cytosine methylation and heat-induced deamination. Biosci Rep. 1986 Apr;6(4):387–393. doi: 10.1007/BF01116426. [DOI] [PubMed] [Google Scholar]
  10. Ehrlich M., Zhang X. Y., Inamdar N. M. Spontaneous deamination of cytosine and 5-methylcytosine residues in DNA and replacement of 5-methylcytosine residues with cytosine residues. Mutat Res. 1990 May;238(3):277–286. doi: 10.1016/0165-1110(90)90019-8. [DOI] [PubMed] [Google Scholar]
  11. Frederico L. A., Kunkel T. A., Shaw B. R. A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy. Biochemistry. 1990 Mar 13;29(10):2532–2537. doi: 10.1021/bi00462a015. [DOI] [PubMed] [Google Scholar]
  12. Hennecke F., Kolmar H., Bründl K., Fritz H. J. The vsr gene product of E. coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease. Nature. 1991 Oct 24;353(6346):776–778. doi: 10.1038/353776a0. [DOI] [PubMed] [Google Scholar]
  13. Hopkins R., Goodman M. F. Asymmetry in forming 2-aminopurine . hydroxymethylcytosine heteroduplexes; A model giving misincorporation frequencies and rounds of DNA replication from base-pair populations in vivo. J Mol Biol. 1979 Nov 25;135(1):1–22. doi: 10.1016/0022-2836(79)90337-1. [DOI] [PubMed] [Google Scholar]
  14. Jones M., Wagner R., Radman M. Mismatch repair of deaminated 5-methyl-cytosine. J Mol Biol. 1987 Mar 5;194(1):155–159. doi: 10.1016/0022-2836(87)90724-8. [DOI] [PubMed] [Google Scholar]
  15. Jones P. A., Rideout W. M., 3rd, Shen J. C., Spruck C. H., Tsai Y. C. Methylation, mutation and cancer. Bioessays. 1992 Jan;14(1):33–36. doi: 10.1002/bies.950140107. [DOI] [PubMed] [Google Scholar]
  16. Lieb M. Specific mismatch correction in bacteriophage lambda crosses by very short patch repair. Mol Gen Genet. 1983;191(1):118–125. doi: 10.1007/BF00330898. [DOI] [PubMed] [Google Scholar]
  17. Lieb M. Spontaneous mutation at a 5-methylcytosine hotspot is prevented by very short patch (VSP) mismatch repair. Genetics. 1991 May;128(1):23–27. doi: 10.1093/genetics/128.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lindahl T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3649–3653. doi: 10.1073/pnas.71.9.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [DOI] [PubMed] [Google Scholar]
  20. Lindahl T., Ljungquist S., Siegert W., Nyberg B., Sperens B. DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J Biol Chem. 1977 May 25;252(10):3286–3294. [PubMed] [Google Scholar]
  21. Lindahl T., Nyberg B. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry. 1974 Jul 30;13(16):3405–3410. doi: 10.1021/bi00713a035. [DOI] [PubMed] [Google Scholar]
  22. Mendelman L. V., Boosalis M. S., Petruska J., Goodman M. F. Nearest neighbor influences on DNA polymerase insertion fidelity. J Biol Chem. 1989 Aug 25;264(24):14415–14423. [PubMed] [Google Scholar]
  23. Mendelman L. V., Petruska J., Goodman M. F. Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem. 1990 Feb 5;265(4):2338–2346. [PubMed] [Google Scholar]
  24. Miller J. H., Ganem D., Lu P., Schmitz A. Genetic studies of the lac repressor. I. Correlation of mutational sites with specific amino acid residues: construction of a colinear gene-protein map. J Mol Biol. 1977 Jan 15;109(2):275–298. doi: 10.1016/s0022-2836(77)80034-x. [DOI] [PubMed] [Google Scholar]
  25. Modrich P. Methyl-directed DNA mismatch correction. J Biol Chem. 1989 Apr 25;264(12):6597–6600. [PubMed] [Google Scholar]
  26. Murchie A. I., Lilley D. M. Base methylation and local DNA helix stability. Effect on the kinetics of cruciform extrusion. J Mol Biol. 1989 Feb 5;205(3):593–602. doi: 10.1016/0022-2836(89)90228-3. [DOI] [PubMed] [Google Scholar]
  27. Petruska J., Goodman M. F., Boosalis M. S., Sowers L. C., Cheong C., Tinoco I., Jr Comparison between DNA melting thermodynamics and DNA polymerase fidelity. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6252–6256. doi: 10.1073/pnas.85.17.6252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rideout W. M., 3rd, Coetzee G. A., Olumi A. F., Jones P. A. 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science. 1990 Sep 14;249(4974):1288–1290. doi: 10.1126/science.1697983. [DOI] [PubMed] [Google Scholar]
  29. Shapiro R., Klein R. S. The deamination of cytidine and cytosine by acidic buffer solutions. Mutagenic implications. Biochemistry. 1966 Jul;5(7):2358–2362. doi: 10.1021/bi00871a026. [DOI] [PubMed] [Google Scholar]
  30. Shenoy S., Ehrlich K. C., Ehrlich M. Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes. J Mol Biol. 1987 Oct 20;197(4):617–626. doi: 10.1016/0022-2836(87)90468-2. [DOI] [PubMed] [Google Scholar]
  31. Singer B., Chavez F., Spengler S. J., Kuśmierek J. T., Mendelman L., Goodman M. F. Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate. Biochemistry. 1989 Feb 21;28(4):1478–1483. doi: 10.1021/bi00430a008. [DOI] [PubMed] [Google Scholar]
  32. Sohail A., Lieb M., Dar M., Bhagwat A. S. A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene. J Bacteriol. 1990 Aug;172(8):4214–4221. doi: 10.1128/jb.172.8.4214-4221.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang R. Y., Kuo K. C., Gehrke C. W., Huang L. H., Ehrlich M. Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA. Biochim Biophys Acta. 1982 Jun 30;697(3):371–377. doi: 10.1016/0167-4781(82)90101-4. [DOI] [PubMed] [Google Scholar]
  34. Watanabe S. M., Goodman M. F. Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6429–6433. doi: 10.1073/pnas.79.21.6429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Watanabe S. M., Goodman M. F. On the molecular basis of transition mutations: frequencies of forming 2-aminopurine.cytosine and adenine.cytosine base mispairs in vitro. Proc Natl Acad Sci U S A. 1981 May;78(5):2864–2868. doi: 10.1073/pnas.78.5.2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wiebauer K., Jiricny J. In vitro correction of G.T mispairs to G.C pairs in nuclear extracts from human cells. Nature. 1989 May 18;339(6221):234–236. doi: 10.1038/339234a0. [DOI] [PubMed] [Google Scholar]
  37. Yatagai F., Glickman B. W. Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13. Mutat Res. 1990 Jan;243(1):21–28. doi: 10.1016/0165-7992(90)90118-4. [DOI] [PubMed] [Google Scholar]

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