Collaborative Research: Mechanisms of Termination of Viral DNA Packaging

智力优势：对于许多大型DNA病毒来说，生产新病毒需要使用蛋白质分子马达，该马达由ATP提供的化学能量提供动力，以迫使复制的病毒DNA进入预先形成的空病毒壳，该壳也由蛋白质组成。病毒DNA被复制成一条连续的长链，必须被切割成完整的单位长度的片段，才能为每个新的病毒颗粒提供完整的DNA基因组。包装DNA的蛋白质马达也会将DNA链切断到正确的长度。对于疱疹病毒和许多细菌病毒，DNA在特定位置被切割，以产生具有正确末端的病毒DNA。包装从精确的初始切割开始，这会产生DNA末端，通过电机将DNA末端穿入外壳。一旦完整的病毒DNA被打包，马达核酸酶就会再次精确地切割DNA，以终止DNA打包周期。该项目旨在确定精确进行末端DNA切割的机制。在这些研究中使用的模型病毒被称为“lambda”，它感染大肠杆菌。之所以选择它，是因为它是研究这一机制最有利的系统之一。Lambda有一个特殊的识别位置，毗邻适当的切割位置，以停止包装电机，从而准确地进行终止切割。DNA包装长度越长，终止效率越高。随着外壳的填充，对包装更多DNA的阻力增加，包装速度变慢，电机继续包装需要更多的能量。解释包装程度如何控制终止效率的模型强调(1)外壳填充的程度，(2)所需的包装能量，或(3)包装速度调节终止。在本项目中，将研究这些因素中的每一个，以确定哪些因素控制终止过程。这些研究将通过使用“光学镊子”来促进，这是一种强大的生物物理技术，将单个DNA分子和单个病毒外壳附着在塑料微球上，并用聚焦的激光进行操纵。单个DNA分子的包装可以通过检测微球的运动和作用在它们上的力来测量。DNA的移动速度、包装所需的能量和包装的程度将通过遗传、生化和生物物理扰动来操纵。还将使用遗传和生物化学方法探索假定的稳定壳蛋白和马达蛋白不同区域的作用。该项目将增加对病毒如何移动、切割和包装DNA的理解。更广泛的影响：病毒DNA包装马达不仅在许多病毒的组装中发挥重要作用，而且很可能与许多其他细胞DNA处理酶共享结构特征和机制，包括解旋酶、细菌染色体分离马达和核酸内切酶。将确保为参与的研究生和本科生提供一个强大的跨学科教育环境，整合生物和物理科学的概念和方法。该项目由两名经验丰富的研究人员领导，他们在生物物理学和分子生物学方面具有互补的研究背景，并有对本科生和来自不同背景的学生进行研究指导的良好记录。在该项目的过程中，还将开发新的本科生物物理学课程材料。K-12科学教育的教材将与R.H.舰队科学中心合作开发。该项目由分子和细胞生物科学部的遗传机制组和化学部的生命过程化学计划共同资助。

Intellectual Merit: For many large DNA viruses, the production of new viruses requires use of a protein molecular motor, powered by chemical energy from ATP, to force the replicated viral DNA into the preformed empty viral shell, which is also made of protein. The viral DNA is replicated as a continuous long chain that must be cut into complete, unit-length pieces to provide each new viral particle with a complete DNA genome. The protein motor that packages the DNA also cuts the DNA chain to the correct length. For the herpes viruses and many bacterial viruses, the DNA is cut at specific sites to generate viral DNA having correct ends. Packaging begins with a precise initial cut, which generates the DNA end that is threaded into the shell by the motor. Once a complete viral DNA has been packaged, the motor nuclease again precisely cuts the DNA to terminate the DNA packaging cycle. This project aims to determine the mechanism by which the terminal DNA cut is precisely made. The model virus to be used in these studies is called "lambda," and it infects the bacterium E. coli. It was chosen because it is one of the most favorable systems for investigating this mechanism. Lambda has a special recognition site, adjacent to the proper cut site, to stop the packaging motor, so that the terminating cut is precisely made. The efficiency of termination increases with the length of the packaged DNA. As the shell fills, the resistance to packaging more DNA increases, the velocity of packaging slows, and more energy is required for the motor to continue packaging. Models to explain how termination efficiency is controlled by the extent of packaging emphasize (1) the extent of shell filling, (2) the packaging energy required, or (3) that packaging velocity regulates termination. In this project, each of these factors will be studied to determine which controls the termination process. These studies will be facilitated by the use of "optical tweezers," a powerful, biophysical technique in which single DNA molecules and individual virus shells are attached to plastic microspheres and manipulated with focused laser beams. Packaging of a single DNA molecule can be measured by detecting the movement of the microspheres and the forces acting on them. The rate of DNA movement, the energy required for packaging, and the extent of packaging will be manipulated through genetic, biochemical, and biophysical perturbations. The roles of a putative shell-stabilizing protein and of various regions of the motor proteins will also be explored using genetic and biochemical methods. The project will increase understanding of how viruses move, cut and package DNA. Broader Impacts: Viral DNA packaging motors not only play an important role in the assembly of many viruses, but very likely also share structural features and mechanisms with many other cellular, DNA-processing enzymes, including helicases, bacterial chromosome segregation motors, and endonucleases. A strong interdisciplinary educational environment, integrating concepts and methods from the biological and physical sciences, will be ensured for participating graduate and undergraduate students. The project is led by two experienced researchers with complementary research backgrounds in biophysics and molecular biology, and strong records of research mentoring of undergraduates and students from diverse backgrounds. New undergraduate biophysics course materials will also be developed in the course of the project. Educational materials for K-12 science education will be developed in collaboration with the R.H. Fleet Science Center.This project is jointly funded by the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences and by the Chemistry of Life Processes program in the Division of Chemistry.