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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM107329-01A1
Application #
8760509
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Willis, Kristine Amalee
Project Start
2014-09-01
Project End
2018-07-31
Budget Start
2014-09-01
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10016
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Sedlyarova, Nadezda; Shamovsky, Ilya; Bharati, Binod K et al. (2016) sRNA-Mediated Control of Transcription Termination in E. coli. Cell 167:111-121.e13
Kamarthapu, Venu; Epshtein, Vitaly; Benjamin, Bradley et al. (2016) ppGpp couples transcription to DNA repair in E. coli. Science 352:993-6
Epshtein, Vitaliy (2015) UvrD helicase: an old dog with a new trick: how one step backward leads to many steps forward. Bioessays 37:12-9
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
Kamarthapu, Venu; Nudler, Evgeny (2015) Rethinking transcription coupled DNA repair. Curr Opin Microbiol 24:15-20