Project Summary/Abstract The long-term goal of this project is to understand how DNA lesions are recognized and repaired in the actively transcribed genome. Harmful DNA lesions, caused by endogenous and environmental agents, must be promptly recognized and repaired in order to avoid deleterious threats to genome integrity. Transcription- coupled nucleotide excision repair (TC-NER) is an important DNA repair pathway as it removes DNA lesions within the transcribed genome. However, little is known about the molecular mechanism of eukaryotic TC-NER initiation. Cockayne Syndrome B protein (CSB), a master TC-NER coordinator, is recruited to the DNA lesion- arrested Pol II site and plays a key role in the initiation of eukaryotic TC-NER. Previously, we reported the first yeast Pol II-Rad26/CSB ternary complex structure, shedding new lights on this important process. However, there is still a fundamental knowledge gap in understanding what happens after CSB recruitment to the DNA lesion-arrested Pol II. Several long-standing questions in the field remain unanswered. First, how does CSB use its DNA translocase activity to remodel the DNA lesion-arrested Pol II and switch Pol II from the transcription elongation mode to the repair mode that leads to the initiation of TC-NER? Second, how is the DNA lesion-arrested Pol II moved away from the DNA lesion to allow the access of repair proteins during TC- NER initiation? Third, are there any missing TC-NER factors that remain to be discovered? If so, how do they fit into this decades-old puzzle? The objective of this proposal is to address these key mechanistic questions in TC-NER initiation. We propose to tackle these challenging questions with an innovative hybrid approach that combines X-ray crystallography, Cryo-EM, computational biology, biochemistry, genetic, and genomic methods. We hypothesize that CSB plays important roles in remodeling lesion-arrested Pol II and coordinates the displacement of elongation factors and Pol II with other repair factors to promote downstream lesion verification steps during the initiation of TC-NER. To test this hypothesis, we propose to investigate the functional interplays between lesion-arrested Pol II complex, Rad26/CSB, and other transcription/repair factors. We propose to elucidate the molecular basis of the enigmatic mechanism of TC-NER initiation. We expect to determine key protein complexes involved in the initiation of TC-NER. Our project has three Specific Aims: Aim 1: Determine the molecular basis of the interplay between Rad26/CSB, Spt4/5, and the DNA lesion- arrested Pol II complex. Aim 2: Elucidate the role of Elf1 in the initiation of TC-NER. Aim 3: Investigate how the lesion-arrested Pol II is displaced during TC-NER initiation. The proposed research is significant and groundbreaking because novel knowledge and structures obtained from this proposal will have a transformative impact on the field of DNA repair field. Ultimately, such knowledge will provide ...