College of Veterinary Medicine
Maintaining genomic integrity is imperative for all living things. Genomes are constantly under threat by endogenous and exogenous sources of DNA damage. RNA polymerase (RNAP) has emerged as a critical player in the sensing of DNA lesions and initiating DNA repair processes. RNAP can sense bulky lesions induced by UV exposure and stimulate homologous recombination. In prokaryotes, transcription and translation are coupled. Translation of a nascent mRNA begins as soon as the ribosome binding site (RBS) emerges from the transcribing RNAP. The physical tethering has a profound impact on RNAP. Coupled ribosomes can restart stalled RNAPs and control their processivity. However, how coupling impacts DNA repair is unknown.
The long-term goal of this project is to determine the impact of transcription-translation coupling on genomic integrity. Preliminary data suggest that translation impedes repair of double-stranded DNA breaks (DSBs) at protein coding genes. Using the Gram-positive model organism Bacillus subtilis, these researchers have found that slowing translation using low doses of translation inhibitors reduces sensitivity to genotoxic agents. Additionally, they find that increasing ribosome stalling by methylation or the ribosome or removal of ribosome processivity factors, also improves survival to genotoxic agents by several orders of magnitude. They predict that slowing translation decouples it from transcription, and that this promotes the ability of RNAP to promote DNA repair of DSBs at protein coding genes. The researchers are investigating this by developing engineered, conditional DSB systems. To accomplish this, they will utilize both homing endonucleases and two different CRISPR nucleases. They will leverage these systems to interrogate DSB repair at all non-essential, protein coding genes in B. subtilis. Specifically, they will use these systems to determine how translation impacts the kinetics of DSB repair, how translation regulates repair proteins at DSBs, and how the translation regulatory mechanisms modulate DSB repair.
The three fundamental processes of transcription, translation, and DNA repair are highly conserved among prokaryotes. These studies will elucidate how these processes are integrated in living cells. This work will not only greatly expand the view of the regulation of DNA repair, but also investigate the molecular mechanisms of transcription-translation coupling in living cells. Transcription, translation, and DNA repair are crucial processes for many aspects of cellular life and, as such, the results of these experiments will provide mechanistic insights into genomic instability in prokaryotes, antimicrobial resistance, and evolution.