CAREER: Mapping Protein Interactions that Link DNA Replication and Nucleosome Assembly

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Genes are compacted and organized in the nucleus of a cell in a precise manner to keep inactive genes “off” and active genes “on.” Aberrant changes in these on/off states result in genomic instability and ultimately affect gene expression in offspring. Maintenance of gene expression relies on several proteins that function together to condense DNA immediately after it is synthesized. This research aims to understand the function and three-dimensional structure of these proteins and how their functions are coordinated to maintain inactive regions of the genome. Another major goal of this project is to create a comprehensive learning environment for young scientists. College students will be heavily involved in performing all experimentation throughout the calendar year and students from local high schools will also have opportunities to participate in research projects every summer. Students will attend weekly scientific development workshops on campus and present their research results to other scientists and the public. The activities proposed are intended to inspire these young students’ interest in science and prepare them for successful future careers.The eukaryotic genome is organized into nucleosomes immediately following DNA replication in a process called replication-coupled nucleosome assembly, which is mediated by two key proteins: chromatin assembly factor 1 (CAF-1) and proliferating cell nuclear antigen (PCNA). The project will employ kinetic, thermodynamic, and structural approaches to understand how PCNA regulates CAF-1 activity. Techniques such as isothermal titration calorimetry and surface plasmon resonance will be used to quantitate interactions between these proteins, and a combination of methods such as X-ray crystallography and small-angle X-ray scattering will be used to determine the structures of complexes formed. The completion of these studies will lead to a greater understanding of the link between DNA replication and nucleosome assembly and how disruptions in this regulation lead to aberrant gene expression.This project is jointly funded by the Genetic Mechanisms program of the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate and the Established Program to Stimulate Competitive Research (EPSCoR).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.