Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 May 1;90(9):3983–3987. doi: 10.1073/pnas.90.9.3983

Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.

M K Dosanjh 1, E L Loechler 1, B Singer 1
PMCID: PMC46430  PMID: 8483914

Abstract

The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.

Full text

PDF
3983

Images in this article

3984Fig. 2
on p.3984
3985Fig. 4
on p.3985
3985Fig. 5
on p.3985

Selected References

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

  1. Aquilina G., Giammarioli A. M., Zijno A., Di Muccio A., Dogliotti E., Bignami M. Tolerance to O6-methylguanine and 6-thioguanine cytotoxic effects: a cross-resistant phenotype in N-methylnitrosourea-resistant Chinese hamster ovary cells. Cancer Res. 1990 Jul 15;50(14):4248–4253. [PubMed] [Google Scholar]
  2. Basu A. K., Essigmann J. M. Site-specifically alkylated oligodeoxynucleotides: probes for mutagenesis, DNA repair and the structural effects of DNA damage. Mutat Res. 1990 Nov-Dec;233(1-2):189–201. doi: 10.1016/0027-5107(90)90162-w. [DOI] [PubMed] [Google Scholar]
  3. Bignami M., Karran P., Lane D. P. Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication. Biochemistry. 1991 Mar 19;30(11):2857–2863. doi: 10.1021/bi00225a018. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brennand J., Margison G. P. Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6292–6296. doi: 10.1073/pnas.83.17.6292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Demple B., Jacobsson A., Olsson M., Robins P., Lindahl T. Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase. J Biol Chem. 1982 Nov 25;257(22):13776–13780. [PubMed] [Google Scholar]
  7. 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]
  8. Dosanjh M. K., Singer B., Essigmann J. M. Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli. Biochemistry. 1991 Jul 16;30(28):7027–7033. doi: 10.1021/bi00242a031. [DOI] [PubMed] [Google Scholar]
  9. Dunn W. C., Tano K., Horesovsky G. J., Preston R. J., Mitra S. The role of O6-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells. Carcinogenesis. 1991 Jan;12(1):83–89. doi: 10.1093/carcin/12.1.83. [DOI] [PubMed] [Google Scholar]
  10. Engel J. D., von Hippel P. H. Effects of methylation on the stability of nucleic acid conformations: studies at the monomer level. Biochemistry. 1974 Sep 24;13(20):4143–4158. doi: 10.1021/bi00717a013. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Loechler E. L. Adduct-induced base-shifts: a mechanism by which the adducts of bulky carcinogens might induce mutations. Biopolymers. 1989 May;28(5):909–927. doi: 10.1002/bip.360280502. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Parthasarathy R., Fridey S. M. Conformation of O6-alkylguanosines: molecular mechanism of mutagenesis. Carcinogenesis. 1986 Feb;7(2):221–227. doi: 10.1093/carcin/7.2.221. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Rebeck G. W., Samson L. Increased spontaneous mutation and alkylation sensitivity of Escherichia coli strains lacking the ogt O6-methylguanine DNA repair methyltransferase. J Bacteriol. 1991 Mar;173(6):2068–2076. doi: 10.1128/jb.173.6.2068-2076.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Samson L., Derfler B., Waldstein E. A. Suppression of human DNA alkylation-repair defects by Escherichia coli DNA-repair genes. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5607–5610. doi: 10.1073/pnas.83.15.5607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Singer B., Chavez F., Goodman M. F., Essigmann J. M., Dosanjh M. K. Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8271–8274. doi: 10.1073/pnas.86.21.8271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Singer B., Essigmann J. M. Site-specific mutagenesis: retrospective and prospective. Carcinogenesis. 1991 Jun;12(6):949–955. doi: 10.1093/carcin/12.6.949. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Xiao W., Derfler B., Chen J., Samson L. Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene. EMBO J. 1991 Aug;10(8):2179–2186. doi: 10.1002/j.1460-2075.1991.tb07753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES