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

在这个项目中，PI将测试DNA修复蛋白利用沿着DNA的一维扩散来定位和识别真核细胞中的光损伤的假设。PI将通过在活细胞中成像荧光标记的蛋白质来研究DNA修复蛋白的募集。PI将在含有多染色体的活细胞中成像蛋白质动力学，其中与DNA相关的蛋白质可以从光学上与染色质间空间中的蛋白质区分开来。此外，PI将通过应用一种通过可见光的多光子吸收来创建三维定位的、类似紫外线的DNA光损伤的方法来触发蛋白质招募。具体的研究目标是：1。表征由可见光的多光子吸收产生的局部DNA光蚀的分布和修复。2. 研究GFP-TopI与活细胞中受损和未受损DNA的相互作用，以确定非特异性关联是否有助于其招募受损DNA。3. 研究XPC和DDB1与活细胞中受损和未受损DNA的相互作用，以确定非特异性关联在这些蛋白质募集到受损DNA中的重要性。PI将根据他在荧光成像方面的研究开发一套实践活动，并在达勒姆公立中学实施这些活动。这些活动的目标是激发年轻学生的科学好奇心，其中大多数是代表性不足的少数群体成员。PI还将继续引进大学预科生和大学生，通过实习的方式进行研究。该项目由物理系的生命系统物理学项目和分子与细胞生物科学系的遗传机制项目共同支持。

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.