This proposal focuses on elucidation of biochemical, physiological, and structural aspects of bacterial RNA polymerase role as a genome-wide DNA damage surveillance vehicle and resolution of its collisions with replisome. Retrograde movement of RNA polymerase, known is backtracking, has been shown to play crucial role in both processes. We have identified several important RNA polymerase-associated factors, particularly UvrD helicase and GreA/B cleavage factors, that stimulate or antagonize backtracking, and have tremendous impact on cell resistance to genotoxic substances. Here we propose a comprehensive set of biochemical, biophysical, genetic, genomics, and structural proteomics experiments aiming at in-depth investigation of all aspects, from molecular to cellular, of this spectrum of transcription-related phenomena.
This proposal aims at illuminating structural, mechanistic and physiological aspects of transcription-replication conflicts and the role RNA polymerase and associated factors (UvrD, Mfd, GreA/B, etc.) play in genome surveillance and repair. We have discovered that RNA polymerase serves as a genome-wide sensor of DNA damage, and that its ability to backtrack from the damage site is essential for base-excision and nucleotide- excision repair (BER and NER, respectively) mechanisms, where it is augmented or antagonized by various factors. Broad evolutionary conservation (bona fide orthology or functional analogy) of the main DNA damage and repair pathways among all cellular organisms makes proposed research relevant to studies of genome stability, mutagenesis and genotoxic substances, from bacteria to humans.
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