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

职业：活细胞中局部 DNA 光损伤修复蛋白募集的时空动态

• 批准号: 1150017
• 负责人: John Papanikolas
• 金额: $ 78.01万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1150017&HistoricalAwards=false
• 关键词: CAREER; Spatiotemporal; Dynamics; Repair; Protein

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.

在这个项目中，PI将测试DNA修复蛋白使用沿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将通过成像活细胞中荧光标记的蛋白质来研究DNA修复蛋白质的募集。PI将成像含有多线染色体的活细胞中的蛋白质动态，其中与DNA相关的蛋白质可以从光学上与染色质间隙中的蛋白质区分开来。此外，PI将通过应用一种开发的方法来触发蛋白质招募，该方法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。具体的研究目标是：1.表征可见光多光子吸收产生的局部DNA光刻的分布和修复。2.研究GFP-TOPI与活细胞中受损和未受损DNA的相互作用，以确定非特异性结合是否有助于其对受损DNA的募集。3.研究XPC和DDB1与活细胞中受损和未受损DNA的相互作用，以确定非特异性结合在这些蛋白与受损DNA募集中的重要性。PI将根据他在荧光成像方面的研究开发一系列实践活动，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对科学的好奇心，他们中的大多数是代表不足的少数群体的成员。PI还将继续通过实习介绍大学预科和本科生进行研究。该项目由物理系的生命系统物理学项目和分子和细胞生物科学系的遗传机制项目共同支持。