Mechanistic investigation of inhibition and dynamics of nucleotide excision repair proteins

Despite being one of the most extensively studied DNA repair pathways, nucleotide excision repair (NER) continues to raise more questions than it answers. Each mechanistic insight reveals new layers of complexity, ensuring that NER remains an active area of research more than six decades after its discovery. This thesis sets out to elucidate key mechanistic and regulatory features of bacterial and eukaryotic NER, with the aim of identifying novel therapeutic opportunities.

A highly conserved putative allosteric pocket BP1 in bacterial UvrA ATPase site was experimentally confirmed by site-directed mutagenesis. In silico screening against BP1 identified riboflavin, a ubiquitous metabolite, as a putative ligand. Subsequent in vitro and in vivo studies confirmed that riboflavin binds to and inhibits UvrA's ATPase and DNA-binding activities, establishing a foundation for potential UvrA metabolic regulation. Furthermore, riboflavin significantly sensitised cells to psoralen-UVA treatment, a dermatological phototherapy that uses psoralen, a photoactive DNA damaging agent, suggesting potential therapeutic applications in dermatology and phototherapy.

In the eukaryotic context, platinum-based chemotherapy remains the predominant anticancer modality. Although NER is the primary repair mechanism for platinum-induced DNA damage, it has not been effectively targeted in cancer therapy. In particular, core Transcription Factor II H (TFIIH) helicase XPD plays a central role in lesion recognition, yet remains pharmacologically overlooked. Virtual screening of XPD's ATPase site identified AR-C118925XX, a P2Y2 receptor antagonist, as a potent ATPase and helicase inhibitor - the first documented small molecule to target XPD.

Detailed interactions of XPD with DNA and lesions remain poorly understood. To address these gaps, a three-bead assay was developed, enabling high spatiotemporal resolution observation of XPD interactions with various DNA substrates and co-factors. Upon validation, this platform will enable the study of XPD in unprecedented detail. To support this study, a custom, disposable microfluidic system was developed for the Lumicks C-Trap optical tweezers. In conclusion, this thesis advances our understanding of NER by revealing novel regulatory mechanisms and identifying the first XPD inhibitor, laying the foundation for NER-targeted therapies in cancer and infectious disease.