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.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Willis, Kristine Amalee
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New York University
Schools of Medicine
New York
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Svetlov, Vladimir; Nudler, Evgeny (2018) Reading of the non-template DNA by transcription elongation factors. Mol Microbiol 109:417-421
Epshtein, Vitaly; Kamarthapu, Venu; Nudler, Evgeny (2017) Strategies and Methods of Transcription-Coupled Repair Studies In Vitro and In Vivo. Methods Enzymol 591:287-306
Sedlyarova, Nadezda; Rescheneder, Philipp; Magán, Andrés et al. (2017) Natural RNA Polymerase Aptamers Regulate Transcription in E. coli. Mol Cell 67:30-43.e6
Demo, Gabriel; Rasouly, Aviram; Vasilyev, Nikita et al. (2017) Structure of RNA polymerase bound to ribosomal 30S subunit. Elife 6:
Rasouly, Aviram; Pani, Bibhusita; Nudler, Evgeny (2017) A Magic Spot in Genome Maintenance. Trends Genet 33:58-67
Pani, Bibhusita; Nudler, Evgeny (2017) Mechanistic insights into transcription coupled DNA repair. DNA Repair (Amst) 56:42-50
Sedlyarova, Nadezda; Shamovsky, Ilya; Bharati, Binod K et al. (2016) sRNA-Mediated Control of Transcription Termination in E. coli. Cell 167:111-121.e13
Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya et al. (2016) Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing. Nature 534:693-6
Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley et al. (2016) ppGpp couples transcription to DNA repair in E. coli. Science 352:993-6
Mejia, Yara X; Nudler, Evgeny; Bustamante, Carlos (2015) Trigger loop folding determines transcription rate of Escherichia coli's RNA polymerase. Proc Natl Acad Sci U S A 112:743-8

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