DNA damage recognition by nucleotide excision repair proteins

描述(申请人提供)：核苷酸切除修复(NER)是一种高度保守的从细菌到人类的途径，可以移除由紫外线和空气污染物等环境因素造成的各种DNA损伤。此外，NER对顺铂等几种抗癌药物诱导的加合物的清除也很重要。DNA修复领域的一个基本问题是，少量的修复蛋白如何扫描数百万(对于细菌)到几十亿个碱基对(对于哺乳动物细胞)的未受损DNA，以找到罕见的受损碱基。该项目将单分子方法(原子力显微镜和斜角荧光)与生化方法相结合，研究细菌和真核核苷酸切除修复蛋白如何检测和移除DNA中受损的核苷酸。这项研究使用了一种新的光学平台来实时观察DNA上的单分子运动，并将提供这些蛋白质机器如何在DNA上组装并追踪DNA损伤的动态视图。这个极具创新性的项目有三个主要目标：1)研究细菌NER蛋白如何实现对DNA损伤的高度特异性识别和修复；2)表征人类损伤识别蛋白XPC-HR23B、XPA、RPA和UV-DDB使用的搜索机制；以及3)检测人类XPD(ERCC2)和XPB(ERCC3)解旋酶蛋白在DNA上的动力学。该项目将测试细菌和人类NER蛋白具有相似的DNA结合模式和损伤检测和处理的搜索机制的假设。完成这些目标将有助于通过开发新的成像技术，在修复的所有阶段直接可视化和实时测量蛋白质复合体，从而为DNA修复领域带来革命性的变化。他们还将开始讨论如何在染色质的背景下检测到损伤。在未来几年，他们还将为在活细胞中实时成像单分子奠定基础。

DESCRIPTION (provided by applicant): Nucleotide excision repair (NER) is a highly conserved pathway from bacteria to humans that removes a wide variety of DNA lesions caused by environmental agents such as UV light and air pollutants. In addition, NER is important for the removal of adducts induced by several anticancer drugs, such as cisplatin. One of the fundamental questions in the field of DNA repair is how a modest number of repair proteins scan through several million (for bacteria) to a few billion base-pairs (for mammalian cells) of non-damaged DNA to find rare damaged bases. This project combines single molecule approaches (atomic force microscopy, and oblique angle fluorescence) with biochemical approaches to examine how bacterial and eukaryotic nucleotide excision repair proteins detect and remove damaged nucleotides from DNA. This study uses a novel optical platform for viewing single molecules in real-time moving on DNA and will give a dynamic view of how these protein machines assemble on DNA and track down DNA lesions. This highly innovative project has three main aims: 1) to investigate how bacterial NER proteins achieve highly specific recognition and repair of DNA damage; 2) to characterize the search mechanisms employed by human damage recognition proteins, XPC-HR23B, XPA, RPA, and UV-DDB; and 3) to examine the dynamics of human XPD (ERCC2), and XPB (ERCC3) helicase proteins on DNA. This project will test the hypothesis that the bacterial and human NER proteins share similar modes of DNA binding and searching mechanisms for damage detection and processing. Completion of these aims will help to revolutionize the field of DNA repair by developing new imaging techniques that allow direct visualization and real-time measurements of protein complexes in all stages of repair. They will also begin to address how damage is detected in the context of chromatin. In future years, they will also lay the ground work for imaging single-molecules in real time in living cells.