Collaborative Research: Mechanisms of Termination of Viral DNA Packaging

合作研究：病毒DNA包装终止机制

• 批准号: 1158328
• 负责人: Douglas Smith
• 金额: $ 58.48万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158328&HistoricalAwards=false
• 关键词: Collaborative; Research; Mechanisms; Termination; Viral

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.

智力优势：对于许多大型DNA病毒来说，新病毒的产生需要使用蛋白质分子马达，由ATP的化学能提供动力，迫使复制的病毒DNA进入预先形成的空病毒壳，这也是由蛋白质组成的。病毒DNA被复制成一个连续的长链，必须被切割成完整的单位长度的片段，以便为每个新的病毒颗粒提供完整的DNA基因组。包裹DNA的蛋白质马达也将DNA链切割到正确的长度。对于疱疹病毒和许多细菌病毒，DNA在特定的位点被切割，以产生具有正确末端的病毒DNA。包装开始于一个精确的初始切割，它产生DNA末端，由马达插入外壳。一旦一个完整的病毒DNA被包装好，马达核酸酶就会再次精确地切割DNA以终止DNA包装循环。该项目旨在确定末端DNA切割精确产生的机制。在这些研究中使用的模型病毒被称为“lambda”，它会感染大肠杆菌。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点，在合适的切割位点附近，停止包装电机，从而精确地进行终止切割。终止的效率随着包装DNA的长度而增加。随着外壳的填充，包装更多DNA的阻力增加，包装速度减慢，电机继续包装需要更多的能量。解释终止效率如何受包装程度控制的模型强调(1)外壳填充程度，(2)包装所需能量，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。“光学镊子”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，并用聚焦的激光束进行操纵。单个DNA分子的包装可以通过检测微球的运动和作用在微球上的力来测量。DNA移动的速度、包装所需的能量和包装的程度将通过遗传、生化和生物物理的扰动来操纵。假定的壳稳定蛋白和马达蛋白的各个区域的作用也将使用遗传和生化方法进行探索。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加工酶（包括解旋酶、细菌染色体分离马达和内切酶）具有相同的结构特征和机制。将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。该项目由两位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有着互补的研究背景，并有丰富的本科生和不同背景学生的研究指导记录。本项目还将开发新的本科生物物理学课程教材。K-12科学教育的教材将与R.H.舰队科学中心合作开发。本项目由分子和细胞生物科学部的遗传机制集群和化学部的生命过程化学项目共同资助。