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 G→T 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 O6POB-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.
Bibliographical noteFunding Information:
We thank Professor Lisa Peterson (University of Minnesota Cancer Center) for the generous gift of O6-POB-dG-containing DNA oligomers. This research was partially supported by a seed grant from the Academic Health Center (University of Minnesota).