Damage Sensing by the Bacterial Nucleotide Excision Repair Pathway

智力优势。双链DNA是细胞的信息储存库，很容易被破坏。细胞已经进化出几种途径，对这种损伤的存在产生生化反应。其中之一是核苷酸切除修复（NER）途径，其特征在于处理化学多样性损伤的能力。在细菌中，NER的第一步由三种蛋白质执行：UvrA，UvrB和UvrC。大量的工作已经描述了介导NER的组件以及它们如何操作的细节。UvrA/UvrB集合体识别损伤并与UvrC和其他因子合作以恢复原始序列。这种大的~300 kDa UvrA/UvrB损伤传感器和处理器的结构是未知的，这阻碍了实验来了解其作用机制。这个项目的目标是获得一个基于结构的核苷酸切除修复的理解。具体目标是解决以下问题：1）UvrA如何利用ATP来感知受损的DNA？（2）UvrA和UvrB如何协同处理DNA损伤？这项工作扩展了UvrA的结构和UvrA/UvrB合奏的第一次视图。了解NER通路的结构和功能非常重要，因为它在维持完整的遗传信息以抵御损伤方面发挥着核心作用，并且在DNA复制、基因表达、进化和细胞过程（如凋亡、衰老和肿瘤发生）中发挥着重要作用。研究者开发了一个综合的多学科教学和研究计划，在生物化学，细胞生物学和结构生物学针对大三，大四和博士生。讲座材料包括当前和过去的研究元素。此外，该研究员还在哈佛大学生物化学专业担任本科生导师。一大批有才华的本科生在研究实验室接受了培训。该科学是跨学科的，涉及分子生物学，生物化学，生物物理学和结构方法。研究结果发表在同行评审的期刊上。研究材料以多种方式提供给科学界，包括蛋白质数据库中的沉积物。

Intellectual Merits. Duplex DNA, the information repository of the cell, is easily damaged. Cells have evolved several pathways that mount biochemical responses to the presence of such damage. One of these is the nucleotide excision repair (NER) pathway, which is distinguished by the ability to process a chemically diverse set of damage. In bacteria, the first steps in NER are performed by three proteins: UvrA, UvrB, and UvrC. A large body of work has described the components that mediate NER and details of how they operate. The UvrA/UvrB ensemble recognizes damage and cooperates with UvrC and other factors to restore the original sequence. The structure of this large ~300 kDa UvrA/UvrB damage sensor and processor is unknown and this has stymied experiments to understand its mechanism of action. The goal of this project is to obtain a structure-based understanding of nucleotide excision repair. The specific aims address the following questions: 1) How does UvrA employ ATP to sense damaged DNA? and 2) How do UvrA and UvrB cooperate to process DNA damage? This work extends on the structure of UvrA and the first-ever view of the UvrA/UvrB ensemble. Understanding the structure and function of the NER pathway is important because of its central role in maintaining the integrety genetic information against damage and its role in DNA replication, gene expression, evolution and cellular processes such as apoptosis, senescence and tumorigenesis.Broader Impacts. The investigator developed an integrated multi-disciplinary teaching and research program in biochemistry, cell biology and structural biology targeting juniors, seniors and doctoral students. Lecture materials include elements from current and past research. In addition, the investigator tutors undergraduate biochemistry majors at Harvard. A large group of talented undergraduates has trained in the investigators laboratory. The science is interdisciplinary and involves molecular biological, biochemical, biophysical, and structural approaches. Results are published in peer-reviewed journals. Research materials are made available to the scientific community in several ways, including depositions in the Protein Data Bank.