NSF-BSF: Defining the relationship between DNA replication kinetics and macromolecular protein assembly at the centromere

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The inheritance of genetic information requires that DNA be replicated and packaged into a form with molecular "adaptors" or "handles" that allow the DNA to be efficiently segregated into daughter cells. However, little is known about the relationship between these two steps of inheritance. This project will lay the foundation of scientific principles governing the coordination of DNA replication and these early steps of its subsequent segregation during cell division. This work will also contribute new understandings of how these DNA metabolic processes occur in real-time within a living cell. The project will result in the interdisciplinary education of graduate students in Israel and the US, cross-training them to critically evaluate experimental techniques in cell biology, mathematics, statistics, and thermodynamics. To further broaden the impact of the project, the Principal Investigator and members of the research groups will contribute to several outreach events to convey the complexity and elegance of DNA inheritance to the general public. PI Bloom will mentor in the Kenan Fellows Program for Teacher leadership, a well-established STEM teacher leadership program in North Carolina. To facilitate broader participation of underrepresented groups in science, the PI will mentor in the Science and Math Achievement and Resourcefulness Track (SMART) program sponsored by the NC-Louis Stokes Alliance for Minority Participation (NC-LSAMP) in science. Centromeres are essential for kinetochore assembly and chromosome segregation during the eukaryotic cell division. At each cell cycle, the complex of centromere DNA and kinetochore proteins must be disassembled to allow DNA replication and then reassembled to allow microtubule attachment and chromosome segregation. Despite extensive studies, little is known regarding the interplay between replisome progression through centromeric regions and the assembly of functional kinetochores at these sites. This project will combine genetics, cell biology, live-cell microscopy, and computational methods to study the relationship between DNA replication, centromere establishment, kinetochore assembly, and chromosome segregation during mitosis. Specifically, to investigate the importance of DNA configurations to centromere assembly, computer simulations and recently developed live-cell imaging approaches for monitoring replication fork progression through active and inactive centromeric regions will be used. This integrative approach, applied to a variety of mutant strains and conditions, will enable the project to shed new light on different aspects of centromeric DNA replication, genome stability, chromosome segregation and cell division. This will provide critical insight into how transcription of centromeric regions affects kinetochore assembly.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.