Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Aug 15;25(16):3354–3361. doi: 10.1093/nar/25.16.3354

Structure of the hydrogen bonding complex of O6-methylguanine with cytosine and thymine during DNA replication.

T E Spratt 1, D E Levy 1
PMCID: PMC146896  PMID: 9241252

Abstract

During DNA replication, mutations occur when an incorrect dNTP is incorporated opposite a carcinogen-modified nucleotide. We have probed the structures of the interaction between O 6-methylguanine ( O 6mG) and cytosine and thymine during replication by kinetic means in order to examine the structure during the rate determining step. The kinetics of incorporation of dCTP and dTTP opposite O 6mG and three analogs, S 6-methyl-6-thioguanine, O 6-methyl-1-deazaguanine and O 6-methylhypoxanthine, have been measured with four polymerases, the Klenow fragment of DNA polymerase I, the Klenow fragment with the proof-reading exonuclease inactivated, Taq and Tth polymerases. In the insertion of dTTP opposite O 6mG, a large decrease in V max/ K m was observed only upon modification of the N1 position. This result is consistent with a Watson-Crick type configuration. For the incorporation of dCTP, the V max/ K m was significantly decreased only with removal of the exocyclic amino group at the 2 position. The pH dependence of the ratio of incorporation of dCTP and dTTP was independent of pH at physiological pH. This result suggests that dCTP is incorporated via an uncharged complex such as the wobble configuration.

Full Text

The Full Text of this article is available as a PDF (130.9 KB).

Selected References

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

  1. Beard W. A., Osheroff W. P., Prasad R., Sawaya M. R., Jaju M., Wood T. G., Kraut J., Kunkel T. A., Wilson S. H. Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta. J Biol Chem. 1996 May 24;271(21):12141–12144. doi: 10.1074/jbc.271.21.12141. [DOI] [PubMed] [Google Scholar]
  2. Bhanot O. S., Ray A. The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7348–7352. doi: 10.1073/pnas.83.19.7348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chambers R. W., Sledziewska-Gojska E., Hirani-Hojatti S., Borowy-Borowski H. uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7173–7177. doi: 10.1073/pnas.82.21.7173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Donlin M. J., Johnson K. A. Mutants affecting nucleotide recognition by T7 DNA polymerase. Biochemistry. 1994 Dec 13;33(49):14908–14917. doi: 10.1021/bi00253a030. [DOI] [PubMed] [Google Scholar]
  5. Dosanjh M. K., Galeros G., Goodman M. F., Singer B. Kinetics of extension of O6-methylguanine paired with cytosine or thymine in defined oligonucleotide sequences. Biochemistry. 1991 Dec 10;30(49):11595–11599. doi: 10.1021/bi00113a015. [DOI] [PubMed] [Google Scholar]
  6. Echols H., Goodman M. F. Fidelity mechanisms in DNA replication. Annu Rev Biochem. 1991;60:477–511. doi: 10.1146/annurev.bi.60.070191.002401. [DOI] [PubMed] [Google Scholar]
  7. Ellison K. S., Dogliotti E., Connors T. D., Basu A. K., Essigmann J. M. Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8620–8624. doi: 10.1073/pnas.86.22.8620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eom S. H., Wang J., Steitz T. A. Structure of Taq polymerase with DNA at the polymerase active site. Nature. 1996 Jul 18;382(6588):278–281. doi: 10.1038/382278a0. [DOI] [PubMed] [Google Scholar]
  9. Frey M. W., Sowers L. C., Millar D. P., Benkovic S. J. The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. Biochemistry. 1995 Jul 18;34(28):9185–9192. doi: 10.1021/bi00028a031. [DOI] [PubMed] [Google Scholar]
  10. Gaffney B. L., Jones R. A. Thermodynamic comparison of the base pairs formed by the carcinogenic lesion O6-methylguanine with reference both to Watson-Crick pairs and to mismatched pairs. Biochemistry. 1989 Jul 11;28(14):5881–5889. doi: 10.1021/bi00440a026. [DOI] [PubMed] [Google Scholar]
  11. Georgiadis P., Smith C. A., Swann P. F. Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras. Cancer Res. 1991 Nov 1;51(21):5843–5850. [PubMed] [Google Scholar]
  12. Georgiadis P., Xu Y. Z., Swann P. F. Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility. Biochemistry. 1991 Dec 17;30(50):11725–11732. doi: 10.1021/bi00114a017. [DOI] [PubMed] [Google Scholar]
  13. Ginell S. L., Kuzmich S., Jones R. A., Berman H. M. Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine. Biochemistry. 1990 Nov 20;29(46):10461–10465. doi: 10.1021/bi00498a005. [DOI] [PubMed] [Google Scholar]
  14. Goodman M. F., Creighton S., Bloom L. B., Petruska J. Biochemical basis of DNA replication fidelity. Crit Rev Biochem Mol Biol. 1993;28(2):83–126. doi: 10.3109/10409239309086792. [DOI] [PubMed] [Google Scholar]
  15. Hill-Perkins M., Jones M. D., Karran P. Site-specific mutagenesis in vivo by single methylated or deaminated purine bases. Mutat Res. 1986 Sep;162(2):153–163. doi: 10.1016/0027-5107(86)90081-3. [DOI] [PubMed] [Google Scholar]
  16. Johnson K. A. Conformational coupling in DNA polymerase fidelity. Annu Rev Biochem. 1993;62:685–713. doi: 10.1146/annurev.bi.62.070193.003345. [DOI] [PubMed] [Google Scholar]
  17. Kalnik M. W., Li B. F., Swann P. F., Patel D. J. O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.C pairing in dodecanucleotide duplexes. Biochemistry. 1989 Jul 25;28(15):6182–6192. doi: 10.1021/bi00441a009. [DOI] [PubMed] [Google Scholar]
  18. Kalnik M. W., Li B. F., Swann P. F., Patel D. J. O6-ethylguanine carcinogenic lesions in DNA: an NMR study of O6etG.T pairing in dodecanucleotide duplexes. Biochemistry. 1989 Jul 25;28(15):6170–6181. doi: 10.1021/bi00441a008. [DOI] [PubMed] [Google Scholar]
  19. Leonard G. A., Thomson J., Watson W. P., Brown T. High-resolution structure of a mutagenic lesion in DNA. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9573–9576. doi: 10.1073/pnas.87.24.9573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Loechler E. L., Green C. L., Essigmann J. M. In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6271–6275. doi: 10.1073/pnas.81.20.6271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loechler E. L. Rotation about the C6-O6 bond in O6-methylguanine: the syn and anti conformers can be of similar energies in duplex DNA as estimated by molecular modeling techniques. Carcinogenesis. 1991 Sep;12(9):1693–1699. doi: 10.1093/carcin/12.9.1693. [DOI] [PubMed] [Google Scholar]
  22. Loveless A. Possible relevance of O-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature. 1969 Jul 12;223(5202):206–207. doi: 10.1038/223206a0. [DOI] [PubMed] [Google Scholar]
  23. Newman P. C., Williams D. M., Cosstick R., Seela F., Connolly B. A. Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC). Biochemistry. 1990 Oct 23;29(42):9902–9910. doi: 10.1021/bi00494a021. [DOI] [PubMed] [Google Scholar]
  24. Patel D. J., Shapiro L., Kozlowski S. A., Gaffney B. L., Jones R. A. Structural studies of the O6meG.C interaction in the d(C-G-C-G-A-A-T-T-C-O6meG-C-G) duplex. Biochemistry. 1986 Mar 11;25(5):1027–1036. doi: 10.1021/bi00353a012. [DOI] [PubMed] [Google Scholar]
  25. Patel D. J., Shapiro L., Kozlowski S. A., Gaffney B. L., Jones R. A. Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex. Biochemistry. 1986 Mar 11;25(5):1036–1042. doi: 10.1021/bi00353a013. [DOI] [PubMed] [Google Scholar]
  26. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [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. Petruska J., Sowers L. C., Goodman M. F. Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1559–1562. doi: 10.1073/pnas.83.6.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rappaport H. P. Replication of the base pair 6-thioguanine/5-methyl-2-pyrimidine with the large Klenow fragment of Escherichia coli DNA polymerase I. Biochemistry. 1993 Mar 30;32(12):3047–3057. doi: 10.1021/bi00063a016. [DOI] [PubMed] [Google Scholar]
  30. Rappaport H. P. The 6-thioguanine/5-methyl-2-pyrimidinone base pair. Nucleic Acids Res. 1988 Aug 11;16(15):7253–7267. doi: 10.1093/nar/16.15.7253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Singer B., Kuśmierek J. T., Folkman W., Chavez F., Dosanjh M. K. Evidence for the mutagenic potential of the vinyl chloride induced adduct, N2, 3-etheno-deoxyguanosine, using a site-directed kinetic assay. Carcinogenesis. 1991 Apr;12(4):745–747. doi: 10.1093/carcin/12.4.745. [DOI] [PubMed] [Google Scholar]
  32. Snow E. T., Foote R. S., Mitra S. Base-pairing properties of O6-methylguanine in template DNA during in vitro DNA replication. J Biol Chem. 1984 Jul 10;259(13):8095–8100. [PubMed] [Google Scholar]
  33. Spratt T. E., Campbell C. R. Synthesis of oligodeoxynucleotides containing analogs of O6-methylguanine and reaction with O6-alkylguanine-DNA alkyltransferase. Biochemistry. 1994 Sep 20;33(37):11364–11371. doi: 10.1021/bi00203a035. [DOI] [PubMed] [Google Scholar]
  34. Spratt T. E., de los Santos H. Reaction of O6-alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that the oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer. Biochemistry. 1992 Apr 14;31(14):3688–3694. doi: 10.1021/bi00129a018. [DOI] [PubMed] [Google Scholar]
  35. Sriram M., van der Marel G. A., Roelen H. L., van Boom J. H., Wang A. H. Structural consequences of a carcinogenic alkylation lesion on DNA: effect of O6-ethylguanine on the molecular structure of the d(CGC[e6G]AATTCGCG)-netropsin complex. Biochemistry. 1992 Dec 1;31(47):11823–11834. doi: 10.1021/bi00162a022. [DOI] [PubMed] [Google Scholar]
  36. Strobel S. A., Cech T. R., Usman N., Beigelman L. The 2,6-diaminopurine riboside.5-methylisocytidine wobble base pair: an isoenergetic substitution for the study of G.U pairs in RNA. Biochemistry. 1994 Nov 22;33(46):13824–13835. doi: 10.1021/bi00250a037. [DOI] [PubMed] [Google Scholar]
  37. Tan H. B., Swann P. F., Chance E. M. Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond. Biochemistry. 1994 May 3;33(17):5335–5346. doi: 10.1021/bi00183a042. [DOI] [PubMed] [Google Scholar]
  38. Waters T. R., Connolly B. A. Interaction of the restriction endonuclease EcoRV with the deoxyguanosine and deoxycytidine bases in its recognition sequence. Biochemistry. 1994 Feb 22;33(7):1812–1819. doi: 10.1021/bi00173a026. [DOI] [PubMed] [Google Scholar]
  39. Williams L. D., Shaw B. R. Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1779–1783. doi: 10.1073/pnas.84.7.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES