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.
Transcription, DNA repair and other essential biological processes require the interaction of proteins with specific segments of DNA. The anthropomorphism "protein recruitment" that is typically used to describe the concerted binding of proteins to accomplish a specific function conceals significant uncertainty about the underlying physical phenomena and chemical interactions governing the formation of macromolecular complexes. This project probes molecular-level mechanisms by which DNA-binding proteins locate target sites in cells. We primarily focused on the development and application of microscopy-based methods to quantitatively characterize the transport of fluorescent fusion proteins in live cell nuclei. These methods enable us to obtain detailed information about the motion and interactions of specific DNA-binding proteins. We developed the ability to perform and quantitatively analyze fluorescence recovery after photobleaching experiments on optical diffraction-limited, three-dimensional volumes of various samples. We applied this technique to investigate the diffusion dynamics of several proteins in live cells containing polytene chromosomes, in which proteins associated with DNA can be optically distinguished from those in the interchromatin space. We discovered that a tracer protein that does not bind specific targets unconjugated green fluorescent protein) exhibits anomalous diffusion in chromosomal regions, but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in cell nuclei. We then used the same method to investigate the mechanism by which large, multi-subunit protein complexes assemble inside cell nuclei. We demonstrated that subunits of the RNA Polymerase II protein are incorporated into a broad distribution of complexes, with sizes ranging from unincorporated proteins to those that have been predicted for fully assembled gene transcription units. We proposed that the broad distribution of macromolecular species allows for mechanistic flexibility in the assembly of transcription complexes, which has important implications for the recruitment of these complexes to active sites in cells.
