The role of Sen1 in transcription coupled DNA repair (TCR)

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DNA repair plays a vital role maintaining genome stability. This project addresses the mechanism of how Sen1 protein is implicated in transcription coupled DNA repair (TCR) in the model organism budding yeast. Sen1 is a homolog of human senataxin, a protein implicated in ataxia-ocular apraxia-2 (AOA2) and an autosomal dominant form of juvenile amyotrophic lateral sclerosis (ALS4). Given the fact that the DNA repair mechanisms are highly conserved, the findings generated from study of the yeast protein are likely to be applicable to other species including human. In addition to scientific advancement on an important DNA repair mechanism, the project will have a significant impact on education, enabling graduate and undergraduate students to acquire research experience and proficiency in yeast genetics and molecular biology. Relevant new methods and findings from the project will be shared with the scientific community and presented in classrooms, local science fairs and at national and international scientific meetings.The TCR pathway of DNA repair has been known for almost 30 years. However, the underlying mechanism of TCR is still poorly understood because of its complexity, especially in eukaryotic cells. Recent findings suggest that Sen1 plays a more direct role in this pathway than the well-known TCR factor Rad26. This project has 3 objectives that build on this significant finding. Objective 1 is to determine the implications of the interactions of Sen1 with Rad2, a single-stranded DNA endonuclease, and with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II), during TCR. A method that allows unambiguous mapping of direct protein-protein interactions in living yeast cells will be utilized to confirm and systematically define the interactions. Objective 2 is to determine the functionality of the minimal essential region of Sen1 (Sen1-MER) in TCR. In addition to its ATPase-helicase activity and nuclear localization function, the relatively large (~ 900 amino acid residues) Sen1-MER may interact with DNA, nascent RNA and certain proteins. It is likely that Sen1-MER also plays an important role in TCR. TCR-deficient Sen1-MER mutants will be isolated and characterized, and the effects of overexpression of Sen1-MER on TCR will be analyzed. Objective 3 is to determine how Sen1 and Rad26 interplay with TCR repressors to regulate transcription bypass of DNA lesions and modulate stalling and/or disruption of the RNAP II complex at DNA lesions for repair. This comprehensive analysis is expected to provide detailed insights into Sen1 and its function in TCR.