Mass spectrometry based approach to study the kinetics of O6-alkylguanine DNA alkyltransferase-mediated repair of O6-pyridyloxobutyl-2'-deoxyguanosine adducts in DNA

Abstract

O(6)-POB-dG (O(6)-[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, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-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 O(6)-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 O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-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 O(6)-POB-G-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 O(6)-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 O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).

Figure 1

HPLC-ESI⁺-MS/MS methodology employed for quantitative analyses of O⁶-POB-G adducts remaining in DNA following AGT repair.

Figure 2

ESI⁺-MS/MS spectra of O⁶-POB-G (A) and D₄-O⁶-POB-G (B) and proposed structures of the major fragments.

Figure 3

Capillary HPLC-ESI⁺-MS/MS analysis of O⁶-POB-G adducts remaining in DNA following AGT repair reaction. 500 fmol of double-stranded DNA (5′-ACCCGCGTCC[O⁶-POB-G]CGCCATGGCC-3′ and its compliment) was incubated with 400 fmol of AGT protein for 20 seconds, followed by quenching with HCl and spiking with 250 fmol of D₄-O⁶-POB-dG. The resulting mixture was subjected to acid hydrolysis and analyzed by capillary HPLC-ESI⁺-MS/MS. The mass spectrometer was operated in SRM mode by monitoring the transitions m/z 299.09 [M + H⁺] →148.1 [POB⁺], 152.07 [Gua + H⁺] for O⁶-POB-G (A) and m/z 303.09 [M + H⁺]→152.07[D₄-POB⁺], [Gua + H⁺] for D₄-O⁶-POB-G (B).

Figure 4

Validation results for quantitative capillary HPLC-ESI⁺-MS/MS method for O⁶-pobG using isotope dilution with D₄-O⁶-POB-G internal standard. Synthetic O⁶-pob-dG-containing DNA duplex (5′-ACCCGCGTCC[O⁶-POB-G]CGCCATGGCC-3′ and its compliment, 0.1-1pmol) was mixed with the corresponding native DNA duplex (1 pmol), D₄-O⁶-POB-dG internal standard, and acid-inactivated AGT protein (500 fmol)(N=4). The resulting mixtures were subjected to mild acid hydrolysis, and O⁶-POB-G adducts were quantified by capillary HPLC-ESI⁺-MS/MS. The results are expressed as a ratio of HPLC-ESI-MS/MS peak areas corresponding to O⁶-POB-G and D₄-O⁶-POB-G versus the actual O⁶-POB-G|D₄-O⁶-POB-G molar ratio.

Figure 5

Time course for the repair of O⁶-POB-G adducts incorporated into synthetic DNA duplexes (5′-ACCCGCGTCC[O⁶-POB-G]CGCCATGGCC-3′ and its compliment) in the presence of increasing amounts of recombinant human AGT protein (20, 200, 400, or 800 fmol). O⁶-POB-G adducts remaining in DNA following the specified reaction times were quantified by isotope dilution HPLC-ESI⁺-MS/MS. The line was generated by fitting to a single-exponential equation using KaleidaGraph software.

Figure 6

Formation of pyridyloxobutylated AGT protein following incubation with O⁶-POB-dG-containing DNA duplex (5′-ACCCGCGTCC[O⁶-POB-G]CGCCATGGCC-3′ and its compliment): Capillary HPLC-ESI⁺-MS/MS analysis of pyridyloxobutylated AGT active site peptide (G¹³⁶NPVPILIPCHR) (A). Time course for the formation of pyridyloxobutylated AGT protein (B). The line was generated by fitting the data to a single exponential equation using KaleidaGraph software.

Figure 7

AGT repair kinetics for O⁶-POB-dG adducts placed opposite dC or dT in synthetic DNA duplexes (5′-AATAGTATCT[O⁶-POB-G]GAGCC-3′ + strand) containing either C or T opposite the adducts). DNA (500 fmol) was incubated with human recombinant AGT protein (400 fmol) for increasing lengths of time (0-50 seconds). The reactions were stopped by HCl quenching, and unrepaired O⁶-POB-G adducts were quantified by isotope dilution HPLC-ESI⁺-MS/MS. Each data point represents an average of four independent measurements. The curves represent the best fit to a second-order exponential equation that provides the rate of O⁶-POB-dG repair by AGT obtained by the KaleidaGraph software. The error is estimated by determining how closely the second order kinetic equation fits the actual data points.

Scheme 1

AGT repair of O⁶-pyridyloxobutyl-dG adducts in DNA