ABSTRACT Elongating RNA polymerase II (Pol II) can be blocked by a variety of DNA damage. The stalled Pol II prevents passage of other RNA and DNA polymerases and blocks exposure of damage to repair proteins, leading to apoptosis or mutagenesis. To avoid these detrimental outcomes, cells activate several mechanisms, including transcription-coupled nucleotide excision repair (TC-NER), to rescue the stalled Pol II. In human cells, the Cockayne Syndrome B (CSB) protein is believed to bind to damage-stalled Pol II and initiate TC-NER. However, there is a critical gap in knowledge concerning how CSB switches Pol II from elongation to a form amenable to DNA repair. Additionally, TC-NER is best known to repair helix-distorting (bulky) DNA lesions, but whether it also repairs non-bulky base damage that occurs more frequently in living cells is poorly understood. To address these important questions, we developed genome-wide and single-nucleotide resolution sequencing methods to map DNA lesions, including bulky cyclobutane pyrimidine dimers (CPDs; UV damage) and non-bulky N-methylpurines (NMPs; alkylation damage). Notably, our CPD-seq data indicates that yeast Rad26, an ortholog of CSB, functions in displacing the transcription elongation factor, Spt4-Spt5, from the stalled Pol II. This function of Rad26 is mainly required for gene coding regions downstream of the first (+1) nucleosome. The eviction of Spt4-Spt5 likely disrupts the closed conformation of the Pol II complex, thereby switching Pol II from elongation to repair. Furthermore, we identified a subset of genes in which TC-NER across the entire coding region was independent of Rad26, suggesting both Rad26-dependent and independent TC-NER mechanisms function in the yeast genome. How CSB functions and whether CSB- independent genes exist in human cells are unclear. In this proposal, we will utilize an improved CPD-seq method to generate genome-wide TC-NER profiles in CSB-proficient and deficient human cells. Comparison of the two repair maps will help identify CSB-dependent and independent genes and guide investigation into their underlying mechanisms (Aim 1). The binding of CSB to Pol II is the first step for TC-NER. Damage removal requires the assembly of a large nucleotide excision repair (NER) complex on DNA. TFIIH is the next crucial NER factor following CSB. Aim 2 will elucidate TFIIH recruitment to test the hypothesis that CSB-mediated Spt4-Spt5 displacement leads to TFIIH binding to the stalled Pol II. Moreover, our NMP-seq data indicates that TC-NER also repairs non-bulky alkylation lesions, suggesting TC-NER targets a broader spectrum of DNA damage than currently appreciated. Aim 3 is based on this intriguing finding and will focus on TC-NER of oxidative base damage, which is the most frequent endogenous damage with a profound role in cancer mutagenesis. Hence, these proposed studies will use innovative approaches and significantly improve TC- NER research by offering genome-wide insights ...