The molecular mechanisms of the Mre11/Rad50 DNA repair complex

本项目的研究目标是详细了解Mre11/Rad50 (MR) DNA修复复合体是如何进行和调节其酶活性的。MR复合物参与DNA双链断裂（DSBs）的加工，这是最有害的DNA损伤形式之一，其修复不当可能导致细胞功能障碍或死亡。该项目的完成有望为生物体如何在面对内部和外部DNA损伤剂的情况下保持其遗传物质的多代提供见解。该项目还将为高中生、本科生和研究生提供高质量的培训经验。PI积极参与多个实习项目，目的是增加选择科学作为职业的人才的多样性。PI正在与爱荷华州立大学的科学绑定项目合作，并将为来访的高中生创建一个实验室模块，涉及紫外线诱导的DNA损伤和修复。最后，PI正在努力通过开发和教授为非科学专业设计的入门科学讲座和实验课程来提高科学素养。本研究项目的总体目标是确定MR复合物的催化和调控机制。先前在PI实验室进行的研究强烈表明，在MR复合体中运作的潜在机制在同源物中很好地保守。研究发现，噬菌体T4 MR复合物的核酸酶活性可以根据所使用的二价阳离子的性质而改变。虽然Mn2+支持强大的3‘到5’ dsDNA外切酶活性，但Mg2+可能支持更生理相关的核酸酶活性。为了确定Mre11在体内发挥其活性所使用的二价阳离子的身份，我们将进行一系列的体内和体外实验。PI提出Rad50螺旋结构域在环状和平行构象状态之间交替，这是由ATP水解循环驱动的，并用于调节ATP酶和核酸酶的活性。结合生物物理技术报告的构象和动态的线圈线圈域将被用来测试这一假设。线圈所采用的整个构象态的集合及其相互转换的速率将被确定。类似的方法将用于监测Mre11的构象变化，并确定这些变化是如何由Rad50的活性控制的。这些研究的结果有望显著提高我们对MR复合体及其在DSB修复过程中如何协调活动的理解。

The research goal of this project is to achieve a detailed understanding of how the Mre11/Rad50 (MR) DNA repair complex carries out and regulates its enzymatic activity. The MR complex is involved in the processing of DNA double-strand breaks (DSBs), which are among the most harmful forms of DNA damage and their improper repair may lead to cellular dysfunction or death. The completion of this project is expected to provide insights into how organisms maintain their genetic material over many generations in the face of internal and external DNA damaging agents. This project will also provide high quality training experiences for high school, undergraduate, and graduate students. The PI actively participates in several internship programs with the goal of increasing the diversity of talented individuals choosing science as a career. The PI is partnering with the Science Bound program at Iowa State University and will create a laboratory module for visiting high school students that involves UV-induced DNA damage and repair. Finally, the PI is working to increase scientific literacy by developing and teaching introductory science lecture and laboratory courses designed for non-science majors. The overall objective of this research project is to define the catalytic and regulatory mechanisms of the MR complex. Previous studies carried out in the PI's lab strongly suggest that the underlying mechanisms that operate in the MR complex are well conserved among homologs. It was found that the nuclease activity of the bacteriophage T4 MR complex can be altered depending on the nature of the divalent cation used. While Mn2+ supports a robust 3' to 5' dsDNA exonuclease activity, Mg2+ may support a more physiologically relevant nuclease activity. A series of in vivo and in vitro experiments will be carried out with the goal of determining the identity of the divalent cation used by Mre11 to perform its activity in vivo. The PI has proposed that the Rad50 coiled-coil domain alternates between ring-like and parallel conformational states, which are driven by the ATP hydrolysis cycle and serve to regulate the ATPase and nuclease activities of the complex. A combination of biophysical techniques that report on the conformation and dynamics of the coiled-coil domain will be employed to test this hypothesis. The entire ensemble of conformational states that the coiled-coil adopts and their rates of interconversion will be determined. Similar methods will be used to monitor conformational changes in Mre11 and to determine how these are controlled by the activity of Rad50. The results of these studies are expected to significantly enhance our understanding of the MR complex and how its activities are coordinated during DSB repair.