PROJECT SUMMARY Nucleotide excision repair (NER) is a crucial DNA repair pathway that removes bulky and helical-distorting lesions (e.g., UV damage) from the genome. NER has two subpathways: Transcription Coupled NER (TC- NER), which repairs damage on the transcribed strand when RNA polymerase II (RNA Pol II) is stalled by the damage, and Global Genomic NER (GG-NER), which repairs damage everywhere else in the genome when damage is recognized by XPC/UV-DDB proteins. After damage recognition, these subpathways converge with the recruitment of Transcription Factor IIH (TFIIH), a 10-subunit protein complex responsible for unwinding the DNA strands to allow DNA incision on the damaged strand. The DNA unwinding function is conducted by XPD, a DNA helicase in TFIIH, with the assistance of other subunits. Mutations of the XPD gene are associated with two genetic disorders: Xeroderma Pigmentosum (XP) and Xeroderma Pigmentosum in combination with Cockayne Syndrome (XP/CS). XP is characterized by extreme sensitivity to UV light and predisposition to skin cancer. XP/CS is characterized by neurodegeneration and predisposition to skin cancer. While the relationship between XPD and human genetic disorders has long been established, why mutations in the same gene cause clinically distinct phenotypes is currently unclear. The objective of this F31 proposal is to determine DNA repair defects caused by XPD mutations, thereby providing mechanistic insights into XP and XP/CS symptoms associated with the mutated XPD gene. We have identified a difference between XP and XP/CS mutants in the overall repair capacity of UV damage using yeast as the model system. This led to my hypothesis that XPD mutations associated with XP or XP/CS differentially affect GG- and TC- NER. To test this hypothesis, Aim 1 will use cyclobutane pyrimidine dimer sequencing (CPD-seq), a genome- wide and strand-specific UV damage sequencing technique, to identify defects in GG- and TC-NER in XPD mutant cells. Repair defects in GG- and TC-NER could be due to impairment in TFIIH’s DNA helicase activity. In Aim 2, I will purify TFIIH protein complex and conduct in vitro biochemical assays to investigate how each XPD mutation impacts TFIIH’s helicase function in DNA unwinding. The neurodegeneration symptom in CS patients is linked with insufficient repair of endogenous small base damage (e.g., oxidative and alkylation); however, the involvement of TFIIH in this repair pathway has not been analyzed previously. In Aim 3 I will determine the impact of XPD mutations on the repair of non-helix-distorting alkylation damage using N- methylpurine sequencing (NMP-seq), a novel sequencing technique developed by our lab. Overall, I will utilize multidisciplinary approaches, including genomics, bioinformatics, and biochemistry to tackle a long-standing question in the DNA repair field. Completion of this project will provide new insights into the mechanism for NER-associated human diseases.