3'-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

Abstract

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. O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine (O(6)-POB-dG). O(6)-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 O(6)-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 O(6)-POB-dG lesion. Conformational analysis and molecular dynamics simulations of DNA containing O(6)-POB-dG suggest that the observed resistance of the O(6)-POB-dG lesion to SVPDE-catalyzed hydrolysis may result from the structural changes in the DNA strand induced by the O(6)-POB group, including C3'-endo sugar puckering and the loss of stacking interaction between the pyridyloxobutylated guanine and its flanking bases. In contrast, O(6)-methylguanine lesion used as a control does not induce similar structural changes in DNA and does not prevent its digestion by SVPDE.

O6‐Alkylguanine DNA Alkyltransferase Repair Activity Towards Intrastrand Cross‐Linked DNA is Influenced by the Internucleotide Linkage

“…Further characterization by enzymatic digestion followed by RP‐HPLC showed the appearance of a new peak with retentions of 9.2 min and 14.7 min for the butylene and heptylene 2′‐deoxyguanosine adducts, respectively (see the Supporting Information). The retention of these dimers was significantly lower compared with those observed for the enzymatic digestion of GG4 and GG7 (16.4 min and 24.8 min), respectively, suggesting incomplete digestion near the cross‐linked site, as previously observed for other DNA modifications …”

“…The retention of these dimers was significantly lower compared with those observed for the enzymatic digestion of GG4 and GG7 (16.4 mina nd 24.8 min), [28] respectively,s uggesting incomplete digestion near the cross-linked site, as previously observed for other DNA modifications. [33,34] UV thermaldenaturation and circular dichroisms tudies of IaCL DNA…”

Synthesis and Characterization of Oligodeoxynucleotides Containing Naphthyridine:Imidazopyridopyrimidine Base Pairs at their Sticky Ends. Application as Thermally Stabilized Decoy Molecules

“…From these results, it was found that incorporation of Na‐N O and Im‐O N units into ODNs improved the resistance to SVPD digestion with an increasing number of these units despite their possessing a natural phosphodiester linkage. Thus far, a number of chemically modified ODNs have been developed, and their SVPD resistance has been discussed 11. The resistance of these ODNs to SVPD digestion has been attributed to a variety of factors, including steric hindrance, interference with the metal ion binding in the exonuclease active site, and altered conformation of the phosphate‐sugar backbone.…”

Quantitation of Pyridyloxobutyl DNA Adducts of Tobacco-Specific Nitrosamines in Rat Tissue DNA by High-Performance Liquid Chromatography−Electrospray Ionization−Tandem Mass Spectrometry

“…First, a different enzymatic hydrolysis procedure was used in the previous study. The DNA may have been digested incompletely due to resistance of O 6 -POB-dGuo to the enzymatic activity of snake venom phosphodiesterase I (23). Second, the previous study did not use deuterated internal standards for each adduct.…”