TY - JOUR
T1 - Mass spectrometry based approach to study the kinetics of O 6-alkylguanine DNA alkyltransferase-mediated repair of O 6-pyridyloxobutyl-2′-deoxyguanosine adducts in DNA
AU - Kotandeniya, Delshanee
AU - Murphy, Dan
AU - Seneviratne, Uthpala
AU - Guza, Rebecca
AU - Pegg, Anthony
AU - Kanugula, Sreenivas
AU - Tretyakova, Natalia
PY - 2011/11/21
Y1 - 2011/11/21
N2 - 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).
AB - 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).
UR - http://www.scopus.com/inward/record.url?scp=81755164040&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=81755164040&partnerID=8YFLogxK
U2 - 10.1021/tx2002993
DO - 10.1021/tx2002993
M3 - Article
C2 - 21913712
AN - SCOPUS:81755164040
SN - 0893-228X
VL - 24
SP - 1966
EP - 1975
JO - Chemical research in toxicology
JF - Chemical research in toxicology
IS - 11
ER -