Intellectual Merit: Cell division requires the replication of the genomic DNA, accomplished by the replication machinery (the replisome). Breaks in the DNA, the presence of DNA binding proteins, and transcription can block the replisome, leading to conflicts that result in genomic instability, mutations, and a failure to segregate fully replicated genomes. Although it is clear that replication pauses as the result of these conflicts, the fate of the replisome during these interruptions is unknown. The overall goal of this research plan is to understand both active and stalled replisomes through a collaborative, interdisciplinary study that takes advantage of proteomics as well as high-resolution single-cell microscopy and mathematical modeling. This research will quantitatively characterize the dynamics and protein composition of the replisome during replication conflicts in vivo. The project addresses these questions in Bacillus subtilis, a genetically tractable bacterial model system that maintains a key feature of eukaryotic replication: distinct leading and lagging strand polymerases. The research will use a new generation of fluorescence microscopy techniques that exploit the small size of the bacterial cell and can observe, count, and track single proteins in living cells. Specifically, this project will determine the protein composition of active and stalled replisomes, both at the population level and at single-protein, single-fork, and single-cell resolution. Furthermore, the stoichiometry and stability of the replisome components at stalled and active replication forks will be determined. Altogether, this work will lead to a deeper understanding of how the replisome faithfully duplicates the genome, in the face of obstacles, in B. subtilis.
Broader Impacts: This research project will impact and advance our understanding of DNA replication and repair in not only Gram-positive bacteria but universally since many key aspects of these processes are conserved across species. Furthermore, this research will implement an interdisciplinary approach that is tightly integrated with undergraduate education. The PIs will recruit talented middle-school, high-school and undergraduate students, including students from under-represented groups, as well as transfer students from two year colleges, for research internships related to this project. Both PIs are especially dedicated to promoting the success of female and underprivileged students by involving them in research early in their education. Lastly, the PIs are committed to building quantitative imaging infrastructure for the Microbiology Department and to continuing the development of a yearly imaging bootcamp course for training biologists at multiple levels in quantitative imaging techniques.
