New Functional Assays Reveal Previously Unrecognized Roles for Nucleotide Excision Repair in Genome Maintenance and Immune Activity

Abstract

Nucleotide excision repair is well-studied as a critical factor in genome maintenance, protecting the cell from external mutagens presenting on the DNA in the form of bulky adducts. The delicate balance of this pathway stands in protecting healthy cells from potentially disastrous damage while also standing as a target for chemotherapeutics in the fight against cancer. While much is known today about the intricate workings of this pathway, we have identified a notable gap in the understanding of how NER interacts with other pathways, both related to DNA repair and other critical survival and differentiation factors. Understanding how important this pathway is for both standard genome maintenance and cancer therapeutics, we set out to expand the field of knowledge around the intricacies of NER in human health and disease. This thesis is divided into three studies. To begin, we identify previously uncharacterized changes in NER capacity induced by mutations in the NEIL1 and XPA proteins. These mutations alter the binding capacity of each protein to other critical factors, decreasing the cell’s NER capacity through pathways previously undescribed. In chapter three, we discuss a new system herein known as FM-NER which allows for simultaneous analysis of the GG-NER and TC-NER sub-pathways. Using this, we identified sub-pathway specific sensitivities in immortalized lines and mutations previously documented in XPA isolated from patient tumors, emphasizing the potential diagnostic promise of this assay. Finally, in chapter four we discuss the identification of notable deficiencies in NER in dendritic cells after their differentiation from monocytes. Using this, we determined that previously documented increases in tumor incidence in XP patients may have a basis in changes in myeloid differentiation.