Project Summary/Abstract The long-term goal of this project is to understand how DNA lesions are recognized and repaired in the transcribed genome. Harmful DNA lesions, caused by endogenous and environmental agents, must be promptly repaired in order to avoid deleterious threats to genome integrity. Transcription-coupled DNA repair (TCR) is an important DNA repair pathway because it removes DNA lesions within the transcribed genome in a transcription-coupled manner. However, the molecular mechanism of eukaryotic TCR remains elusive. There is evidence that Cockayne Syndrome B protein (CSB), a master TCR coordinator, is recruited to the DNA lesion- arrested Pol II site and plays a key role in the initiation of eukaryotic TCR. However, there is a fundamental gap in understanding the molecular basis of CSB recruitment to the DNA lesion-arrested Pol II. A long-standing question in the field is how CSB recognizes and interacts with the DNA lesion-arrested Pol II and subsequently initiates TCR. The objective of this proposal is to elucidate the roles of CSB in TCR initiation. The central hypothesis is that CSB recognizes the structural features of specific Pol II domains and the nucleic acid scaffold in arrested Pol II complexes, undergoes significant conformational changes, and is subject to autoregulation by its own regulatory motifs. This hypothesis has been formulated on the basis of preliminary data produced in the applicants' laboratories and will be tested by performing structural and functional characterizations of the interactions between CSB and the arrested Pol II complex. This approach is innovative, because it utilizes a novel hybrid method that combines X-ray crystallography, Cryo-EM, biophysics, biochemistry, and genetics. 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 and vertically advance the understanding of how transcription-coupled repair is initiated. Ultimately, such knowledge will provide a framework for developing novel TCR targeting therapeutics against cancer and other human diseases.