Damage Sensing by the Bacterial Nucleotide Excision Repair Pathway

智力上的优点。双链DNA是细胞的信息库，很容易受到破坏。细胞已经进化出几种途径，对这种损伤的存在做出生化反应。其中之一是核苷酸切除修复(NER)途径，它的特点是能够处理一系列不同的化学损伤。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调解NER的组件以及它们如何运行的细节。UvrA/UvrB合奏识别损伤，并与UvrC和其他因素合作，恢复原始序列。这种大型~300 kDa UvrA/UvrB损伤传感器和处理器的结构尚不清楚，这阻碍了对其作用机制的实验研究。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。具体目的是解决以下问题：1)UvrA如何利用ATP来检测DNA损伤？2)UvrA和UvrB如何协同处理DNA损伤？这项工作扩展了UvrA的结构和对UvrA/UvrB系综的首次观察。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免受损伤以及在DNA复制、基因表达、进化和细胞过程(如细胞凋亡、衰老和肿瘤发生)中发挥着重要作用。研究人员开发了生物化学、细胞生物学和结构生物学的综合多学科教学和研究计划，目标是大三、大四和博士生。授课材料包括来自当前和过去研究的元素。此外，调查员还指导哈佛大学生物化学专业的本科生。一大批有才华的本科生在调查实验室接受了培训。这门科学是跨学科的，涉及分子生物学、生化、生物物理和结构方法。研究结果发表在同行评议的期刊上。科学界可以通过几种方式获得研究材料，包括蛋白质数据库中的证词。

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.