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

Abstract

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).

Kinetics of O6-Pyridyloxobutyl-2′-deoxyguanosine Repair by Human O6-alkylguanine DNA Alkyltransferase

“…35,36 The activity of the AGT protein was determined by titrating the recombinant protein with DNA duplexes containing site specific O 6 -MeG, followed by HPLC-ESI + -MS/MS analysis as described previously. 31,37 D 4 - O 6 -POB-dG was a gift from Professor Stephen Hecht (University of Minnesota Masonic Cancer Center). HBEC cells were grown in Keratinocyte SFM media (Life Technologies, NY) supplemented with human recombinant Epidermal Growth Factor (EGF 1-53, Life Technologies, NY) and Bovine Pituitary Extract media (Life Technologies, NY).…”

“…O 6 -POB-G and D 4 - O 6 -POB-G were purified by solid phase extraction (SPE) on Strata X cartridges. 31 O 6 -POB-G was quantified by capillary HPLC-ESI + -MS/MS using D 4 - O 6 -POB-G internal standard as described elsewhere. 31 Data reported are an average of 9–14 individual experiments.…”

“…O 6 -POB-G and D 4 - O 6 -POB-G were purified by solid phase extraction (SPE) and quantified by capillary HPLC-ESI + -MS/MS as described previously. 31 …”

“…O 6 -POB-G was quantified by capillary HPLC-ESI + -MS/MS following acid hydrolysis and SPE purification as described elsewhere. 31 …”

N3-Substituted Temozolomide Analogs Overcome Methylguanine-DNA Methyltransferase and Mismatch Repair Precipitating Apoptotic and Autophagic Cancer Cell Death

“…Small molecule inhibitors of poly(ADP-ribose)polymerase-1, a critical BER protein, are yielding promising results clinically, in combination with TMZ and as single-agent chemotherapy in patients whose tumors possess homologous recombination DNA repair defects [13,14]. Another validated, but as yet preclinical protein target, mandatory to BER, is abasic endonuclease-1 (APE-1); in preclinical tests, APE-1 inhibition potentiates TMZ activity [15].…”