Kinetics of O⁶-Methyl-2‘-deoxyguanosine Repair by O⁶-Alkylguanine DNA Alkyltransferase within K-ras Gene-Derived DNA Sequences

Abstract

O(6)-Methyl-2'-deoxyguanosine (O(6)-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O(6)-Me-dG is directly repaired by the specialized DNA repair protein, O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O(6)-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI+-MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O(6)-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8-17 of the K-ras protooncogene, 5'-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3', where G5, G6, G7, G8, G9, G10, or G11 was replaced with O(6)-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G --> A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O(6)-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC-ESI-MS/MS was used to determine the amounts of O(6)-Me-G remaining in DNA. The relative extent of demethylation for O(6)-Me-dG adducts located at G5, G6, G7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O(6)-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5'-G6G7T-3') were similar (1.4 x 10(7) M(-1) s(-1) vs 7.4 x 10(6) M(-1) s(-1), respectively), suggesting that O(6)-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC-ESI-MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.

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

“…By comparison, the rates of repair of O 6 -Me-dG adducts present at the same positions were 1.4 × 10 7 M −1 s −1 and 7.4 × 10 6 M −1 s −1 , respectively. 37 These results indicate that O 6 -POB-dG repair by AGT is much slower than that of O 6 -Me-dG and shows a greater dependence on local sequence environment. This can be explained by steric effects of the bulky pyridyloxobutyl group, which may interfere with correct placement of the adduct within the protein active site, inhibiting alkyl transfer.…”

“…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).…”

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

“…17,18 The catalytic activity of the AGT protein was determined by incubating with known amounts of DNA duplexes containing O 6 -MeG. 19 Approximately 75% of the total protein was active, depending on the aliquot. D 4 - O 6 -POB-dG was a gift from Professor Stephen Hecht (University of Minnesota).…”

“…22-24 Oligonucleotide concentrations were determined from the amounts of 2′-deoxyguanosine present in enzymatic digests using a previously published protocol. 19,25,26 …”

Multifaceted Roles of Alkyltransferase and Related Proteins in DNA Repair, DNA Damage, Resistance to Chemotherapy, and Research Tools

“…There was a slightly slower rate when the O 6 -methylguanine was 3′ to guanine residue (1, 124-126). A somewhat larger effect has been reported for a neighboring 5-methylcytosine but these effects were highly dependent on the overall sequence context (124, 127, 128).…”