Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant <i>O</i>⁶-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an <i>O</i>⁶-Alkylguanine Adductome.

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Abdelhady, Rasha, Senthong, Pattama, Eyers, Claire E ORCID: 0000-0002-3223-5926, Reamtong, Onrapak, Cowley, Elizabeth, Cannizzaro, Luca, Stimpson, Joanna, Cain, Kathleen, Wilkinson, Oliver J, Williams, Nicholas H et al (show 4 more authors) , Barran, Perdita E, Margison, Geoffrey P, Williams, David M and Povey, Andrew C (2023) Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant <i>O</i>⁶-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an <i>O</i>⁶-Alkylguanine Adductome. Chemical research in toxicology, 36 (12). pp. 1921-1929.

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Abstract

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, <i>O</i>⁶-methylguanine <i>O</i>⁶-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA <i>O</i>⁶-alkylguanines (<i>O</i>⁶-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple <i>O</i>⁶-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different <i>O</i>⁶-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known <i>O</i>⁶-MethylG (<i>O</i>⁶-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing <i>S</i>-methyl, <i>S</i>-ethyl, <i>S</i>-propyl, <i>S</i>-hydroxyethyl, <i>S</i>-carboxymethyl, <i>S</i>-benzyl, and <i>S</i>-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding <i>O</i>⁶-alkGs. The LOQ of ASPs containing <i>S</i>-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol <i>O</i>⁶-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing <i>S</i>-methylcysteine at levels that correlated with those of <i>O</i>⁶-MeG determined previously by HPLC-radioimmunoassay (<i>r</i>² = 0.74; p = 0.014). <i>O</i>⁶-CMG, a putative <i>O</i>⁶-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of <i>O</i>⁶-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA <i>O</i>⁶-alkG adductome and, given the mutagenic potential of <i>O</i>⁶-alkGs, can provide mechanistic information about cancer pathogenesis.

Item Type:	Article
Uncontrolled Keywords:	Humans, Colorectal Neoplasms, Cysteine, O(6)-Methylguanine-DNA Methyltransferase, Peptides, DNA, Oligodeoxyribonucleotides, DNA Repair, Catalytic Domain, Mass Spectrometry
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	01 Feb 2024 09:38
Last Modified:	01 Feb 2024 09:38
DOI:	10.1021/acs.chemrestox.3c00207
Open Access URL:	https://doi.org/10.1021/acs.chemrestox.3c00207
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3178239