Kinetics of <i>O</i>⁶‑Pyridyloxobutyl-2′-deoxyguanosine Repair by Human <i>O</i>⁶‑alkylguanine DNA Alkyltransferase

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>⁶-[4-oxo-4-(3-pyridyl)­but-1-yl]­deoxyguanosine (<i>O</i>⁶-POB-dG) lesions. If not repaired, <i>O</i>⁶-POB-dG adducts induce large numbers of G → A and G → T mutations. Previous studies have shown that <i>O</i>⁶-POB-dG can be directly repaired by <i>O</i>⁶-alkylguanine-DNA alkyltransferase (AGT), which transfers the pyridyloxobutyl group from <i>O</i>⁶-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>⁶-POB-dG in duplex DNA. Synthetic oligodeoxynucleotide duplexes containing site-specific <i>O</i>⁶-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⁺-MS/MS analysis of <i>O</i>⁶-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>⁶-POB-dG was 2–7 times slower than that of <i>O</i>⁶-Me-dG adducts. To evaluate the contribution of AGT to <i>O</i>⁶-POB-dG repair in human lung, normal human bronchial epithelial cells (HBEC) were treated with model pyridyloxobutylating agent, and <i>O</i>⁶-POB-dG adduct repair over time was monitored by HPLC-ESI⁺-MS/MS. We found that HBEC cells were capable of removing <i>O</i>⁶-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (<i>O</i>⁶-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>⁶-POB-dG at a specific sequences contributes to mutational spectra observed in smoking-induced lung cancer

DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA–protein cross-links (DPCs). Interstrand DNA–DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human <i>O</i>⁶-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT–DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles