Mechanisms of Viral DNA Packaging

病毒 DNA 包装机制

• 批准号: 7088743
• 负责人: CARLOS Jose BUSTAMANTE
• 金额: $ 53.97万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088743
• 关键词: X ray crystallography; bacterial virus; bacteriophage lambda; chemical kinetics; conformation; cryoelectron microscopy; fluorescence microscopy; fluorescence polarization; fluorescence resonance energy transfer; intracellular transport; ionic bond; nanotechnology; virus DNA; virus genetics; virus infection mechanism; virus replication

DESCRIPTION (provided by applicant): In many viruses, an empty "prohead" is assembled and subsequently filled with DNA by the action of an ATP-dependent portal motor. DNA packaging occurs in many phages, herpesviruses, adenoviruses and poxviruses, and is one of the least understood steps in viral replication. In this remarkable process, the DNA s compacted to near-crystalline density--overcoming energetic penalties due to electrostatic repulsion, DNA bending stiffness, and entropy. A single molecule optical tweezers assay has enabled real time measurement of phage phi29 packaging and shows that the motor exerts forces of up to 60 pN on the DNA, making it one of the strongest molecular motors. DNA packaging in phi29 will be further studied using optical tweezers to determine the fundamental chemical and mechanical steps that generate motor movement, the biochemical kinetics of motor operation, and the nature of the motor-DNA interaction. Biochemical and structural studies suggest that the packaging machine may represent a new class of rotary motors. State-of-the- art single-molecule fluorescence and spectroscopy techniques will be developed to track conformational changes in the motor and test the rotary motor hypothesis. Application of new theoretical and mathematical modeling techniques will yield further insights. Results of biophysical measurements will be integrated with structure determination by X-ray crystallography and cryo-electron microscopy (from established collaborations) and used to guide the development of models. Single molecule studies of phage lambda DNA packaging will test the universality of packaging motors. Knowledge of the motor mechanism will aid the development of anti-viral drugs and yield insights into other DNA translocating enzymes such as polymerases helicases, recombination factors, and chromatin assembly factors. These studies will shed light on fundamental principles of protein-nucleic acid and protein-protein interactions, macromolecular self-assembly and conformational change, and mechanochemical energy transduction.

描述（由申请人提供）：在许多病毒中，空的“前头”被组装，随后通过依赖于ATP的门脉运动的作用被DNA填充。 DNA 包装发生在许多噬菌体、疱疹病毒、腺病毒和痘病毒中，是病毒复制中最不为人所知的步骤之一。在这个非凡的过程中，DNA 被压缩到接近晶体的密度，克服了静电排斥、DNA 弯曲刚度和熵造成的能量损失。单分子光镊检测能够实时测量噬菌体 phi29 包装，并显示该电机对 DNA 施加高达 60 pN 的力，使其成为最强的分子电机之一。将使用光镊进一步研究 phi29 中的 DNA 包装，以确定产生运动运动的基本化学和机械步骤、运动操作的生​​化动力学以及运动与 DNA 相互作用的性质。生化和结构研究表明，包装机可能代表一类新型旋转电机。将开发最先进的单分子荧光和光谱技术来跟踪电机的构象变化并测试旋转电机假设。新的理论和数学建模技术的应用将产生进一步的见解。生物物理测量结果将与 X 射线晶体学和冷冻电子显微镜（来自已建立的合作）的结构确定相结合，并用于指导模型的开发。噬菌体 lambda DNA 包装的单分子研究将测试包装马达的通用性。了解运动机制将有助于抗病毒药物的开发，并深入了解其他 DNA 转位酶，如聚合酶解旋酶、重组因子和染色质组装因子。这些研究将揭示蛋白质-核酸和蛋白质-蛋白质相互作用、大分子自组装和构象变化以及机械化学能量转导的基本原理。