Mechanisms of Viral DNA Packaging

病毒 DNA 包装机制

• 批准号: 8232085
• 负责人: CARLOS Jose BUSTAMANTE
• 金额: $ 48.75万
• 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232085
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Adenoviruses; Affect; Bacteriophages; Base Pairing; Binding; Biochemical; Biological Assay; Biological Models; Capsid; Cell physiology; Characteristics; Chemicals; Collaborations; Complement; Coupling; Cryoelectron Microscopy; Crystallography; DNA; DNA Packaging; Electrostatics; Elements; Entropy; Event; Family member; Generations; Genetic; Genome; Head; Herpesviridae; Hydrolysis; In Vitro; Individual; Investigation; Laboratories; Light; Location; Measurement; Measures; Mechanics; Methods; Modeling; Molecular; Motor; Mutagenesis; Nature; Optics; Pathway interactions; Phase; Positioning Attribute; Process; Production; Prokaryotic Cells; Property; Proteins; Regulation; Relative (related person); Resolution; Role; Rotation; Staging; Structure; System; Tail; Techniques; Time; Torque; Viral; Viral Packaging; Virus; density; drug development; instrument; instrumentation; laser tweezer; model development; mutant; novel; operation; pressure; public health relevance; response; segregation; single molecule; viral DNA

DESCRIPTION (provided by applicant): In many viruses an empty "prohead" is assembled and subsequently filled with DNA by the action of ATP dependent portal motor. DNA packaging occurs in many phages, herpesviruses, adenoviruses and poxvirues and it is therefore an important target for anti-viral drug development. In this application, we propose to use genetic, biochemical and biophysical approaches to expand and deepen our previous single-molecule studies of the packaging process by the portal motor of bacteriophage F29. This phage is an ideal system to investigate this process as a robust in-vitro packaging assay has been available. During the packaging process the DNA is compacted to near-crystalline density-overcoming energetic penalties due to electrostatics repulsion, DNA bending stiffness, and entropy. Because this motor is comprised of a pentameric ring of ASCE ATPases, its study will shed important light into the operation of other members of this family and of the larger superfamily of AAA+ ring ATPases, known to be responsible for a large number of cellular functions, from protein unfolding and degradation to chromosomal segregation in prokaryotes. We propose to characterize in great detail the various chemical and mechanical events during the operational cycle of each ATPase (i.e., ATP binding, hydrolysis, product release, translocation, etc) and to establish the precise timing or coordination among the cycles of the individual subunits in the ring. We will also establish the ability of the motor to generate torque, its magnitude and its generation mechanism relative to the production of linear force. These studies will be complemented by a characterization of the nature and strength of the contacts made between the DNA and the motor and its modulation during the various phases of the mechanochemical cycle. Finally, we will establish the participation of other non-catalytic elements of the motor such as the head-tail connector and the regulation of the motor's dynamics by the internal DNA pressure generated inside the capsid during the packaging process. To carry out these studies we will take advantage of state-of-the-art optical tweezers instrumentation in our laboratory. This instrument will make it possible for us to follow the packaging process with the unprecedented spatial resolution of 1A with a temporal resolution of 1 sec. 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 of this process. PUBLIC HEALTH RELEVANCE: We propose to study the detailed molecular mechanisms of the packaging motor responsible for genome compaction in the bacteriophage 29 using genetic, biochemical and biophysical approaches. Specifically we will use a single molecule optical tweezers approach to characterize the coordination between the individual chemical cycles of the five subunits in this ring ATPase. We will also establish the ability of this motor to generate torques and the nature and strength of its interaction with the DNA. Finally, we wish to investigate how the increasing internal DNA pressure regulates the dynamics of the motor during packaging.

描述（由申请人提供）：在许多病毒中，一个空的“ prohead”组装了，随后通过ATP依赖门户电机的作用填充了DNA。 DNA包装发生在许多噬菌体，疱疹病毒，腺病毒和POXVIRUE中，因此是抗病毒药物发育的重要靶标。在此应用中，我们建议使用遗传，生化和生物物理方法来扩展和加深我们先前通过噬菌体F29的门户电动机对包装过程的单分子研究。此噬菌体是研究此过程的理想系统，因为已经可以使用强大的体外包装测定法。在包装过程中，由于静电排斥，DNA弯曲刚度和熵，DNA被压缩到近乎结晶密度的势损。 Because this motor is comprised of a pentameric ring of ASCE ATPases, its study will shed important light into the operation of other members of this family and of the larger superfamily of AAA+ ring ATPases, known to be responsible for a large number of cellular functions, from protein unfolding and degradation to chromosomal segregation in prokaryotes.我们建议在每个ATPase的操作周期（即ATP结合，水解，水解，产品释放，易位等）中详细表征各种化学和机械事件，并在环中单个亚基的周期之间建立精确的时序或协调。我们还将建立电动机产生扭矩的能力，相对于线性力的产生，其幅度及其产生机制。这些研究将通过在机械化学循环的各个阶段的DNA与电动机之间的接触性质和强度的特征来补充。最后，我们将建立电动机其他非催化元件的参与，例如头尾连接器，以及在包装过程中帽子内产生的内部DNA压力对电动机的动力学调节。为了进行这些研究，我们将利用实验室中最先进的光学镊子仪器。该工具将使我们有可能以1A的前所未有的空间分辨率以1秒的时间分辨率遵循包装过程。生物物理测量结果将与X射线晶体学和冷冻电子显微镜（来自已建立的协作）的结构确定相结合，并用于指导该过程模型的开发。 公共卫生相关性：我们建议使用遗传，生化和生物物理方法研究包装电机的详细分子机制。具体而言，我们将使用单个分子光镊方法来表征该环ATPase中五个亚基的单个化学循环之间的协调。我们还将确定该电动机产生扭矩以及与DNA相互作用的性质和强度的能力。最后，我们希望研究内部DNA压力增加如何调节包装过程中电动机的动力学。