Investigating the catalytic mechanisms of DNA repair enzymes using classical and quantum mechanical computational techniques

Abstract

DNA is continuously subjected to chemical damage arising from endogenous metabolic processes and environmental stressors. If left unrepaired, the resulting DNA lesions can disrupt genome stability and interfere with essential cellular processes. In some cases, damage occurs to bases that also serve regulatory roles, meaning that repair must restore not only the chemical structure of DNA, but also the epigenetic information encoded within the genome. To counteract these threats, cells employ a diverse network of DNA repair enzymes that recognize and process chemically-modified bases. Defects in DNA repair pathways have been linked to mutagenesis, disease development, and altered cellular function. Despite their biological importance, the catalytic mechanisms of many DNA repair enzymes remain poorly understood, with multiple and sometimes conflicting mechanistic proposals present in the literature. In this thesis, computational methodologies including molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were used to clarify and identify atomic-level details of the catalytic mechanisms involved in glycosidic bond cleavage and oxidative DNA repair. Specifically, the monofunctional glycosylase UdgX was investigated, focusing on its unique suicide inactivation mechanism involving the formation of a covalent DNA–protein crosslinked intermediate during uracil excision. Mutational variants were also investigated to determine how changes to the active site environment shift the catalytic pathway from crosslink to hydrolysis. To complement this work, monofunctional alkyladenine glycosylase (AAG) was also studied to better understand how lesion identity influences catalytic competency and determines when repair is supported or inhibited despite the enzyme’s broad substrate specificity. In particular, the understudied etheno lesion 3,N⁴-etheno-5-methylcytosine (ε5mC) was examined alongside the canonical substrates 3,N⁴-ethenocytosine (εC) and 1,N⁶-ethenoadenine (εA). Finally, the oxidative repair enzyme ALKBH2 was investigated to characterize how etheno lesions, including εC and ε5mC, are recognized and repaired through direct reversal repair, and to determine whether chemical modifications to these lesions influences the catalytic mechanism and repair outcome. Together this work provides a comprehensive mechanistic framework for understanding how DNA repair enzyme’s function and reveals how subtle variations in active site architecture and solvation dictate pathway selection at the molecular level.