The AlkB-family enzymes have been shown to oxidize alkylative DNA adducts to different extents dependent upon the pH conditions to which they are subject <i>in vitro</i>. Reduced activity of enzymes such as ABH2 and ABH3 in humans, could contribute to accumulation of the cytotoxic DNA adducts, 1-methyladenine or 3-methylcytosine. This study will investigate the <i>in vitro </i>pH profiles of human ABH2 and ABH3 and E. coli AlkB in regard to their abilities to oxidize 1-methyladenine and 3-methylcytosine in single and double-strand contexts. All synthetic oligonucleotides were synthesized utilizing the Mermade 4 platform, and all AlkB-family enzymes were expressed and purified. The impact of pH conditions on AlkB-mediated oxidation of methylated DNA was quantified by Anion Exchange HPLC. Our findings show that pH can significantly influence the ability of these enzymes to repair DNA damage, and even alter substrate strand preferences as is the case for ABH2 when subject to more acidic pH conditions.

pH effects on oxidative modification of alkylative DNA adducts by the AlkB-family enzymes

ACS Fall 2021

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