Mass Spectrometry Based Approach to Study the Kinetics of O6-Alkylguanine DNA Alkyltransferase Mediated Repair of O6-pyridyloxobutyl-2′-deoxyguanosine Adducts in DNA

摘要

O⁶-POB-dG (O⁶-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O⁶-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O⁶-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O⁶-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O⁶-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI⁺-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O⁶-POB-dG adducts. In our approach, synthetic DNA duplexes containing O⁶-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D4-O⁶-POB-dG internal standard and mild acid hydrolysis to release O⁶-POB-guanine (O⁶-POB-G) and D4-O⁶-POB-guanine (D4-O⁶-POB-G), samples are purified by solid phase extraction (SPE), and O⁶-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI⁺-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O⁶-POB-dG-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O⁶-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI⁺-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O⁶-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5′-AATAGTATCT[O⁶-POB-G]GAGCC-3′) opposite either C or T. Faster rates of alkyl transfer were observed when O⁶-POB-dG was paired with T rather than C (k = 1.74 × 10⁶ M⁻¹s⁻¹ vs 1.17 × 10⁶ M⁻¹s⁻¹).