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，新病毒的生产需要使用由 ATP 化学能提供动力的蛋白质分子马达，迫使复制的病毒 DNA 进入预先形成的空病毒壳，该病毒壳也是由蛋白质制成的。病毒 DNA 作为一条连续的长链进行复制，必须将其切割成完整的单位长度片段，以便为每个新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 链切割成正确的长度。 对于疱疹病毒和许多细菌病毒来说，DNA在特定位点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，切割产生 DNA 末端，并通过电机穿入外壳。 一旦完整的病毒 DNA 被包装，马达核酸酶会再次精确切割 DNA 以终止 DNA 包装周期。 该项目旨在确定精确进行末端 DNA 切割的机制。这些研究中使用的模型病毒称为“lambda”，它会感染大肠杆菌。选择它是因为它是研究该机制最有利的系统之一。 Lambda 有一个特殊的识别点，靠近正确的切割点，可以停止包装电机，从而精确地进行终止切割。 终止效率随着包装 DNA 长度的增加而增加。 随着外壳被填满，包装更多 DNA 的阻力增加，包装速度减慢，电机需要更多能量才能继续包装。 解释终止效率如何通过包装程度控制的模型强调 (1) 壳填充程度，(2) 所需的包装能量，或 (3) 包装速度调节终止。在此项目中，将对每个因素进行研究，以确定哪个因素控制终止过程。 这些研究将通过使用“光镊”来促进，这是一种强大的生物物理技术，其中单个 DNA 分子和单个病毒壳附着在塑料微球上，并用聚焦激光束进行操纵。 单个 DNA 分子的包装可以通过检测微球的运动和作用在其上的力来测量。 DNA 运动的速率、包装所需的能量以及包装的程度将通过遗传、生物化学和生物物理扰动来控制。 假定的壳稳定蛋白和运动蛋白各个区域的作用也将使用遗传和生化方法进行探索。该项目将增进对病毒如何移动、切割和包装 DNA 的了解。 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核酸内切酶）共享结构特征和机制。将为参与的研究生和本科生确保一个强大的跨学科教育环境，融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，并且对来自不同背景的本科生和学生进行了良好的研究指导。该项目过程中还将开发新的本科生生物物理学课程材料。 K-12科学教育教材将与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集群和化学部的生命过程化学项目共同资助。