Kinetics of <i>O</i><sup>6</sup>‑Pyridyloxobutyl-2′-deoxyguanosine
Repair by Human <i>O</i><sup>6</sup>‑alkylguanine
DNA Alkyltransferase
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Abstract
Tobacco-specific
nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) and N-nitrosonicotine (NNN) are potent carcinogens believed
to contribute to the development of lung tumors in smokers. NNK and
NNN are metabolized to DNA-reactive species that form a range of nucleobase
adducts, including bulky <i>O</i><sup>6</sup>-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine
(<i>O</i><sup>6</sup>-POB-dG) lesions. If not repaired, <i>O</i><sup>6</sup>-POB-dG adducts induce large numbers of G →
A and G → T mutations. Previous studies have shown that <i>O</i><sup>6</sup>-POB-dG can be directly repaired by <i>O</i><sup>6</sup>-alkylguanine-DNA alkyltransferase (AGT), which
transfers the pyridyloxobutyl group from <i>O</i><sup>6</sup>-alkylguanines in DNA to an active site cysteine residue within the
protein. In the present study, we investigated the influence of DNA
sequence context and endogenous cytosine methylation on the kinetics
of AGT-dependent repair of <i>O</i><sup>6</sup>-POB-dG in
duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific <i>O</i><sup>6</sup>-POB-dG adducts within <i>K-ras</i> and <i>p53</i> gene-derived DNA sequences were incubated
with recombinant human AGT protein, and the kinetics of POB group
transfer was monitored by isotope dilution HPLC-ESI<sup>+</sup>-MS/MS
analysis of <i>O</i><sup>6</sup>-POB-dG remaining in DNA
over time. We found that the second-order rates of AGT-mediated repair
were influenced by DNA sequence context (10-fold differences) but
were only weakly affected by the methylation status of neighboring
cytosines. Overall, AGT-mediated repair of <i>O</i><sup>6</sup>-POB-dG was 2–7 times slower than that of <i>O</i><sup>6</sup>-Me-dG adducts. To evaluate the contribution of AGT to <i>O</i><sup>6</sup>-POB-dG repair in human lung, normal human
bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating
agent, and <i>O</i><sup>6</sup>-POB-dG adduct repair over
time was monitored by HPLC-ESI<sup>+</sup>-MS/MS. We found that HBEC
cells were capable of removing <i>O</i><sup>6</sup>-POB-dG
lesions, and the repair rates were significantly reduced in the presence
of an AGT inhibitor (<i>O</i><sup>6</sup>-benzylguanine).
Taken together, our results suggest that AGT plays an important role
in protecting human lung against tobacco nitrosamine-mediated DNA
damage and that inefficient AGT repair of <i>O</i><sup>6</sup>-POB-dG at a specific sequences contributes to mutational spectra
observed in smoking-induced lung cancer