DNA damage recognition by nucleotide excision repair proteins

描述（由申请人提供）：核苷酸切除修复（NER）是从细菌到人类的一个高度保守的途径，可以消除由环境因素（如紫外线和空气污染物）引起的各种DNA损伤。此外，NER对于去除一些抗癌药物（如顺铂）诱导的加合物也很重要。DNA修复领域的一个基本问题是，数量不多的修复蛋白如何扫描数百万（对于细菌）到几十亿碱基对（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，研究细菌和真核生物核苷酸切除修复蛋白如何从DNA中检测和去除受损的核苷酸。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上组装和追踪DNA损伤的动态视图。这个高度创新的项目有三个主要目的：1)研究细菌NER蛋白如何实现高度特异性的DNA损伤识别和修复；2)研究人体损伤识别蛋白XPC-HR23B、XPA、RPA和UV-DDB的搜索机制；3)研究人类XPD （ERCC2）和XPB （ERCC3）解旋酶蛋白在DNA上的动态变化。该项目将验证细菌和人类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。这些目标的完成将有助于通过开发新的成像技术来彻底改变DNA修复领域，这些成像技术可以在修复的所有阶段直接可视化和实时测量蛋白质复合物。他们还将开始解决如何在染色质的背景下检测损伤。在未来的几年里，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. PUBLIC HEALTH RELEVANCE: DNA damage recognition by nucleotide excision repair proteins PI: Bennett Van Houten Project Narrative: Nucleotide excision (NER) repair removes damage from our genome that is induced by a wide variety of environmental agents, including UV light and air pollution. Alterations in NER can cause increased mutations, cancer and cell death and manifest in several human disorders including xeroderma pigmentosum, aging and neurodegeneration. This project will gain insight into how these repair proteins detect and remove DNA damage.