The Bacterial Nucleotide Excision Repair Pathway: Structure and Mechanism

智力优势：了解与基因和基因组如何遗传和改变相关的生物化学途径仍然是一个紧迫的优先事项。这项工作的目标是获得对细菌中一种这样的途径的基于结构的理解，例如，核苷酸切除修复（NER）的DNA损伤诱导暴露于紫外线。细菌NER的第一步是由三种蛋白质UvrA、UvrB和UvrC完成的，它们在修复过程中结合成几种动态的多蛋白复合物。UvrA-UvrB系综扫描基因组，将含有损伤的DNA与天然DNA区分开来，然后与UvrC核酸酶和其他因子合作修复损伤。这项工作将测试新的假设三个不完全理解的NER机制：a）歧视的天然损坏，B）ATP/ADP动态NER期间，和c）本地化的Uvr B的病变。本研究采用多学科方法，整合结构，生物化学，单分子和生物物理策略，以了解基因组扫描病变时相关大分子的结构，功能和动力学之间的关系。NER期间基于结构的遗传机制分析将为DNA修复，DNA复制，遗传和转录之间的联系提供更深入的见解。更广泛地说，分析DNA的基于形状的识别之间的关系，由PI提出作为区分受损和天然DNA的机制，和蛋白质构象变化将有助于全面理解生物系统中DNA被蛋白质识别的基本原理。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定量技能。PI通过使用对DNA修复的研究来解决这个问题，DNA修复是一个基本的生物学过程，作为教授这些技能的平台。这个项目建立在PI过去在哈佛大学的努力之上，现在继续在纽约城市学院进行。CCNY是一个公共博士学位-授予机构，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，其中所描述的研究将被整合，并将影响~100名本科生/博士生。此外，与城市议会合作，PI将在纽约公立学校系统开展教育推广活动，包括与学生（6-12年级）会面，并与教师合作制定课程计划。PI小组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，他们也教导和指导初级同事。研究结果将发表在同行评审的期刊上。研究材料将以几种方式提供给科学界，包括向蛋白质数据库提供。

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.