Investigations into DNA Structural Perturbations Induced by Oxidative Lesions and Site-Specific Drug Conjugates

Abstract

DNA integrity is constantly exposed to oxidative stress and reactive metabolites, producing chemical lesions that alter structure and disrupt replication and repair. Understanding these distortions at the molecular level reveals how DNA damage contributes to mutagenesis and drug response. This dissertation examines two representative cases: the oxidative lesion 6-oxo-M1dG and the drug-induced pixantrone-AP conjugate. The DNA lesion M1dG, formed from oxidative base propenals or lipid peroxidation product malondialdehyde (MDA), can oxidize in vivo to 6-oxo-M1dG, which blocks replication and is bypassed mutagenically by polymerase η. To study its structural impact, 6-oxo-M1dG was site-specifically incorporated into a DNA duplex and analyzed by NMR and restrained molecular dynamics. The lesion intercalated into the duplex while maintaining an anti-glycosidic conformation, displacing the complementary cytosine into major groove. Perturbations were localized to the lesion site and adjacent base pairs, causing a 15C decrease in melting temperature, consistent with thermal destabilization. Separately, abasic (AP) sites arise from spontaneous base loss, oxidative damage, enzymatic excision of alkylated, or alkylation of guanine at N7 generate abasic sites. These electrophilic sites can form Schiff-base conjugates with anthracycline drugs such as pixantrone (PIX). NMR and restrained molecular dynamics of fully reduced PIX-AP conjugates revealed two regioisomers (Ra and Rb) intercalating into DNA helix major groove from minor groove, displacing C18. Ra forms more rapidly and intercalates less deeply than Rb. Melting studies showed PIX conjugation stabilizes AP-site duplexes relative to unmodified DNA. Together, these results define the structural consequences of oxidative and drug-induced DNA damage and reveal how pixantrone covalently interacts with abasic DNA, informing its potential mechanism of chemotherapeutic action.