The Bacterial Nucleotide Excision Repair Pathway: Structure and Mechanism

智力价值：了解与基因和基因组如何遗传和改变相关的生化途径仍然是当务之急。这项工作的目标是从结构上了解细菌中的一种这样的途径，例如，紫外线照射引起的DNA损伤的核苷酸切除修复(NER)。细菌NER的第一步是由三种蛋白质UvrA、UvrB和UvrC执行的，随着修复的进行，它们结合成几个动态的多蛋白复合体。UvrA-UvrB组合扫描基因组，区分含有损伤的DNA和天然DNA，然后与UvrC核酸酶和其他因子合作修复损伤。这项工作将检验关于NER三个尚未完全了解的机制的新假说：a)区分天然和受损的NER，b)NER过程中的ATP/ADP动态，以及c)UvrB在病变中的定位。这项研究采取了多学科的方法，整合了结构、生化、单分子和生物物理策略，以了解在扫描基因组是否有病变时相关大分子的结构、功能和动力学之间的关系。对NER过程中遗传机制的基于结构的分析将为DNA修复、DNA复制、遗传和转录之间的联系提供更深层次的见解。更广泛地说，分析基于形状的DNA识别和蛋白质构象变化之间的关系将有助于全面理解生物系统中蛋白质识别DNA的基本原理。更广泛的影响：科学教育的核心挑战之一是如何向年轻同事传授量化技能。PI通过使用DNA修复的研究作为教授此类技能的平台来解决这个问题。DNA修复是一种基本的生物学过程。这个项目建立在PI过去在哈佛大学的努力基础上，现在纽约城市学院继续进行。CCNY是一家公共授予博士学位的机构，吸引来自不同社会经济阶层、民族和种族背景的本科生/博士生。为了推进科学、技术、工程和数学(STEM)教育，PI将在CCNY教授两门课程，所述研究将纳入其中，将影响约100名本科生/博士生。此外，PI将与城市议会合作，在纽约公立学校系统内开展教育宣传，包括与学生(6-12年级)举行会议，并与教师就教案进行合作。在国际和平研究所的小组中进行的科学是多学科的，涉及几个研究小组之间的密切合作。PI积极指导博士后，反过来，他们又教授和指导初级同事。研究结果将发表在同行评议的期刊上。研究材料将以几种方式向科学界提供，包括向蛋白质数据库提供证词。

Intellectual Merit: Understanding the biochemical pathways associated with how genes and genomes are inherited, and changed, remains an urgent priority. The goal of this work is to gain structure-based understanding of one such pathway in bacteria, e.g., nucleotide excision repair (NER) of DNA damage induced by exposure to ultraviolet light. The first steps of bacterial NER are performed by three proteins, UvrA, UvrB, and UvrC, which associate into several dynamic multi-protein complexes as repair proceeds. The UvrA-UvrB ensemble scans the genome, distinguishing lesion-containing damaged DNA from native, and, then, cooperates with the UvrC nuclease and other factors to repair the damage. This work will test novel hypotheses for three incompletely understood mechanisms of NER: a) discrimination of native from damaged, b) ATP/ADP dynamics during NER, and c) localization of UvrB to the lesion. This study takes a multi-disciplinary approach, integrating structural, biochemical, single-molecule, and biophysical strategies to understand relationships between the structure, function, and dynamics of relevant macromolecules as the genome is scanned for lesions. Structure-based analyses of genetic mechanisms during NER will provide deeper insights into the connections between DNA repair, DNA replication, inheritance, and transcription. More broadly, analysis of the relationship between shape-based recognition of DNA, proposed by the PI as a mechanism for discriminating damaged and native DNA, and protein conformational changes will contribute to a comprehensive understanding of fundamental principles that underlie the way that DNA is recognized by proteins in biological systems. Broader Impacts: One of the central challenges for science education is how to impart quantitative skills to younger colleagues. The PI approaches this problem by using research on DNA repair, a fundamental biological process, as a platform to teach such skills. This project builds on the PI's past efforts at Harvard University, which now continue at City College of New York. CCNY is a public Ph.D.-granting institution that attracts undergraduate/doctoral students from diverse socioeconomic strata, and ethnic and racial backgrounds. Towards advancing education in science, technology, engineering and mathematics (STEM), the PI will teach two courses at CCNY, into which the described research will be integrated, and which will impact ~100 undergraduate/doctoral students. In addition, in partnership with The Urban Assembly, the PI will perform educational outreach in the New York Public School system, which will include meetings with students (grades 6-12), and collaborations with teachers on lesson plans. The science performed in the PI's group is multi-disciplinary and involves close collaborations between several research groups. The PI actively mentors post-docs, and, they, in turn, teach and mentor junior colleagues. The results of the research will be published in peer-reviewed journals. Research materials will be made available to the scientific community in several ways, including depositions to the Protein Data Bank.