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DNA damage recognition by nucleotide excision repair proteins

核苷酸切除修复蛋白识别 DNA 损伤

基本信息

批准号：
8580937
负责人：
Bennett Van Houten
金额：
$ 39.93万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-09 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8580937
关键词：
Address Affect Aging Air Pollutants Air Pollution Antineoplastic Agents Atomic Force Microscopy Bacteria Bacterial Proteins Base Pairing Binding Biochemical Cell Death Cells Charge Chromatin Cisplatin Collaborations Color Complex DNA DNA Binding DNA Damage DNA Folding DNA Repair DNA Repair Pathway DNA analysis DNA lesion DNA-Binding Proteins Detection Disease Dissociation ERCC2 gene ERCC3 gene Engineering Excision Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy Future Genome Goals Human Hydrolysis Image Imagery Imaging Techniques Knowledge Label Lead Lesion Life Malignant Neoplasms Mammalian Cell Measurement Mediating Molecular Machines Movement Mutation Neoplastic Cell Transformation Nerve Degeneration Nucleosomes Nucleotide Excision Repair Nucleotides Optics Pathway interactions Plasma Predisposition Process Proteins Quantum Dots Reaction Research Resistance Role Scanning Site Staging Surface Testing Thermodynamics Time UV induced DNA damage Ultraviolet Rays Work XPA gene Xeroderma Pigmentosum Zinc Fingers adduct base cancer cell cytotoxic environmental agent environmental mutagens fluorescence imaging helicase innovation insight mutant novel prevent protein complex public health relevance repaired replication factor A single molecule

项目摘要

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.

描述（由申请人提供）：核苷酸切除修复（NER）是一种从细菌到人类的高度保守途径，可去除由环境因子（如紫外线和空气污染物）引起的各种DNA损伤。此外，NER对于去除几种抗癌药物（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，少量的修复蛋白如何扫描数百万（对于细菌）到数十亿（对于哺乳动物细胞）的未受损DNA碱基对，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）与生物化学方法相结合，以研究细菌和真核生物的核苷酸切除修复蛋白如何检测和去除DNA中受损的核苷酸。这项研究使用了一种新型的光学平台来观察单分子在DNA上的实时移动，并将动态观察这些蛋白质机器如何在DNA上组装并追踪DNA损伤。这个高度创新的项目有三个主要目标：1）研究细菌NER蛋白如何实现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-HR 23 B，XPA，RPA和UV-DDB所采用的搜索机制; 3）研究人类XPD（ERCC 2）和XPB（ERCC 3）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。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，这些技术允许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，他们还将为活细胞中真实的单分子成像奠定基础。