CAREER: Spatiotemporal Dynamics of Repair Protein Recruitment to Localized DNA Photolesions in Live Cells

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In this project the PI will test the hypothesis that DNA repair proteins use one-dimensional diffusion along DNA to locate and identify photo-lesions in eukaryotic cells. The PI will investigate DNA repair protein recruitment by imaging fluorescently labeled proteins in live cells. The PI will image protein dynamics in live cells containing polytene chromosomes, in which proteins associated with DNA can be optically distinguished from those in the inter-chromatin space. In addition the PI will trigger protein recruitment by applying a method developed to create three-dimensionally localized, UV-like DNA photo-lesions via multi-photon absorption of visible light. The specific research objectives are to: 1. Characterize the distribution and repair of local DNA photolesions produced by multiphoton absorption of visible light. 2. Investigate the interactions of GFP-TopI with damaged and undamaged DNA in live cells, in order to determine if nonspecific associations contribute in its recruitment to damaged DNA. 3. Investigate the interactions of XPC and DDB1 with damaged and undamaged DNA in live cells, in order to determine the importance of nonspecific associations in the recruitment of these proteins to damaged DNA. The PI will develop a set of hands-on activities based on his research in fluorescence imaging, and implement these in Durham public middle schools. The goal of these activities is to enliven the scientific curiosity of young students, most of which are members of underrepresented minority groups. The PI will also continue to introduce precollege and undergraduate students to research through internships in the group.This project is being jointly supported by the Physics of Living Systems program in the Division of Physics and the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.