Mechanisms of Viral DNA Packaging

病毒 DNA 包装机制

• 批准号: 8065973
• 负责人: CARLOS Jose BUSTAMANTE
• 金额: $ 50.16万
• 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/8065973
• 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，空的“前头部”被组装，随后通过ATP依赖性门静脉马达的作用被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，因此它是抗病毒药物开发的重要目标。在这个应用中，我们建议使用遗传，生物化学和生物物理的方法来扩展和深化我们以前的单分子研究的包装过程中的门电机的噬菌体F29。该噬菌体是研究该过程的理想系统，因为已经有了稳健的体外包装测定。在包装过程中，DNA被压缩到接近晶体密度，克服了由于静电排斥、DNA弯曲刚度和熵而产生的能量损失。由于该马达由ASCE ATP酶的五聚环组成，因此其研究将为该家族的其他成员和AAA+环ATP酶的更大超家族的操作提供重要的启示，AAA+环ATP酶已知负责大量的细胞功能，从蛋白质解折叠和降解到原核生物中的染色体分离。我们建议非常详细地表征每个ATP酶的操作周期期间的各种化学和机械事件（即，ATP结合、水解、产物释放、易位等），并在环中各个亚基的循环之间建立精确的定时或协调。我们还将建立电动机产生扭矩的能力，其大小及其相对于线性力产生的产生机制。这些研究将通过表征DNA和电机之间的接触的性质和强度及其在机械化学循环的各个阶段的调制来补充。最后，我们将建立参与的其他非催化元件的电机，如头-尾连接器和电机的动力学的调节由内部的DNA压力产生的衣壳在包装过程中。为了进行这些研究，我们将利用我们实验室中最先进的光镊仪器。该仪器将使我们能够以前所未有的1A空间分辨率和1秒的时间分辨率跟踪包装过程。生物物理测量的结果将与X射线晶体学和冷冻电子显微镜（来自已建立的合作）的结构测定相结合，并用于指导该过程模型的开发。 公共卫生关系：我们建议使用遗传学，生物化学和生物物理学的方法来研究负责基因组压缩在噬菌体29的包装电机的详细分子机制。具体来说，我们将使用单分子光镊方法来表征该环ATP酶中五个亚基的各个化学循环之间的协调。我们还将确定这个马达产生扭矩的能力以及它与DNA相互作用的性质和强度。最后，我们希望研究如何增加内部DNA压力调节包装过程中的电动机的动态。