Nucleic Acids Research, 2003, Vol. 31, No. 7 1984-1994
© 2003 Oxford University Press
3'-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine
1 University of Minnesota Cancer Center, 2 Department of Medicinal Chemistry, University of Minnesota School of Pharmacy and 3 Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN 55455, USA
*To whom correspondence should be addressed at 760E CCRB, University of Minnesota Cancer Center, 806 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455, USA. Tel: +1 612 626 3432; Fax: +1 612 626 5135; Email: trety001{at}umn.edu
Tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a chemical carcinogen thought to be involved in the initiation of lung cancer in smokers. NNK is metabolically activated to methylating and pyridyloxobutylating species that form promutagenic adducts with DNA nucleobases, e.g. O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-POB-dG). O6-POB-dG is a strongly mispairing DNA lesion capable of inducing both G
A and GT base changes, suggesting its importance in NNK mutagenesis and carcinogenesis. Our earlier investigations have identified the ability of O6-POB-dG to hinder DNA digestion by snake venom phosphodiesterase (SVPDE), a 3'-exonuclease commonly used for DNA ladder sequencing and as a model enzyme to test nuclease sensitivity of anti-sense oligonucleotide drugs. We now extend our investigation to three other enzymes possessing 3'-exonuclease activity: bacteriophage T4 DNA polymerase, Escherichia coli DNA polymerase I, and E.coli exonuclease III. Our results indicate that, unlike SVPDE, 3'-exonuclease activities of these three enzymes are not blocked by O6-POB-dG lesion. Conformational analysis and molecular dynamics simulations of DNA containing O6-POB-dG suggest that the observed resistance of the O6-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O6-POB group, including C3'-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases. In contrast, O6-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE.