The Molecular Mechanisms of the Mre11/Rad50 DNA Repair Complex

智力价值：这个项目的目标是实现对Mre11/Rad50(MR)DNA修复复合体的机械理解，该复合体参与真核生物、古菌和一些噬菌体中DNA双链断裂(DSB)的处理。双链断裂是最有害的DNA损伤形式之一，其不适当的修复可能会导致严重的染色体重排，导致细胞功能障碍或死亡。MR复合体的双重活性(ATP水解酶和核酸酶)是修复被毒素或蛋白质化学修饰的双链断裂所必需的。本研究项目将集中于定量定义MR复合体的动力学、热力学和构象动力学。本研究的目标是：1)确定RAD50对ATP结合和水解的功能作用和机制；2)确定金属依赖的MR复合核酸酶活性变化背后的机制，并发现额外的蛋白质在DSB切除中的可能作用；3)表征mre11的构象变化，确定mre11二聚体的作用。该项目的完成有望增加对DSB修复的了解，更广泛地说，将增加对生物体如何在多代中保持其基因组完整性的了解。更广泛的影响：该项目涉及与遗传和结构生物学实验室的合作，因此将使本科生和研究生接触到生化、生物物理学和遗传学研究相结合所产生的协同效应。该项目将为高中、本科生和研究生提供高质量的培训机会。除了在PI的国内部门对学生进行多年培训外，PI还参与了女性科学与工程实习计划，并将成为乔治·华盛顿·卡弗实习计划的导师。这些项目的目标是增加选择科学作为职业的人才的多样性。PI还在开发一门课程，将科学新闻专业的学生安排在一个研究实验室，在那里他们将参加小组会议，采访实验室成员，并观察日常活动。本课程的目的是让新闻系学生了解实验结果的获得方式和速度。接受采访和观察的实验室成员也将受益于学会向非专家解释他们的研究。此外，PI正在开发一门结合酶理论、实用酶特性和现代数据拟合/模型判别分析的课程。在整个课程中，PI将使用他自己研究的例子，如果可能，生成讨论数据的学生将向他们的同龄人展示实验。这种对等教学已被发现是一种非常有效的学习工具，对同行教师和同行学生都是如此。这个项目是由分子和细胞生物科学系的遗传机制集群和刺激竞争性研究的实验计划共同资助的。

Intellectual Merit: The goal of this project is to achieve a mechanistic understanding of the Mre11/Rad50 (MR) DNA repair complex, which is involved in the processing of DNA double-strand breaks (DSBs) within eukaryotes, archaea, and some bacteriophage. DSBs are among the most deleterious forms of DNA damage and their improper repair may cause gross chromosomal rearrangements, leading to cellular dysfunction or death. The dual activities of the MR complex (ATP hydrolysis and nuclease) are required for the repair of DSBs that have been chemically modified by toxins or proteins. This research project will focus on defining, in quantitative terms, the kinetics, thermodynamics, and conformational dynamics of the MR complex. The objectives of this research project are to: 1) Define the functional role and mechanism of ATP binding and hydrolysis by Rad50; 2) Determine the mechanism behind the metal-dependent change in MR complex nuclease activity and to discover the possible roles of additional proteins in DSB resection; 3) Characterize the conformational changes in Mre11 and determine the role of Mre11 dimerization. The completion of this project is expected to increase understanding of DSB repair and, more generally, how organisms maintain their genomic integrity through multiple generations. Broader Impacts: This project involves collaborations with both genetic and structural biology laboratories and will therefore expose and educate undergraduate and graduate students to the synergy that results from the combination of biochemical, biophysical, and genetic research. The project will provide high quality training opportunities for high school, undergraduate, and graduate students. In addition to the multi-year training of students in the PI's home department, the PI participates in the Women in Science and Engineering internship program and will become a mentor for the George Washington Carver internship program. The goal of these programs are to increase the diversity of talented individuals choosing science as a career. The PI is also developing a course that places science journalism students in a research laboratory where they will attend group meetings, interview members of the laboratory, and observe daily activities. The objective is for the journalism students to appreciate the manner and pace at which experimental findings are obtained. The laboratory members who are being interviewed and observed will also benefit by learning to explain their research to non-experts. In addition, the PI is developing a course that combines enzyme theory, practical enzyme characterization, and modern data fitting/model discrimination analysis. Throughout the course the PI will use examples from his own research, and when possible, the students that generated the data under discussion will present the experiments to their peers. This type of peer-to-peer teaching has been found to be a very effective learning tool for both the peer-teachers and peer-students.This project is co-funded by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences and by the Experimental Program to Stimulate Competitive Research.