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(申请人提供)：在许多病毒中，一个空的“前头”被组装起来，随后在ATP依赖的门户马达的作用下填充DNA。DNA包装存在于许多噬菌体、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靶点。在这一应用中，我们建议使用遗传、生化和生物物理方法来扩展和深化我们之前通过噬菌体F29的门户马达对包装过程的单分子研究。该噬菌体是研究这一过程的理想系统，因为已经有了一种强大的体外包装试验。在包装过程中，DNA被压实到接近晶体的密度--克服了由于静电斥力、DNA弯曲硬度和熵而造成的能量损失。由于这个马达是由一个五聚体的ASCE ATPase环组成的，它的研究将为这个家族的其他成员以及更大的AAA+环ATPase超家族的运作提供重要的线索，已知的AAA+环ATPase超家族负责从蛋白质的展开和降解到原核生物的染色体分离等大量细胞功能。我们建议更详细地描述每个ATPase工作周期中的各种化学和机械事件(即，ATP结合、水解、产物释放、转位等)，并建立环中各个亚基周期之间的精确定时或协调。我们还将确定电机产生扭矩的能力、其大小及其相对于线性力产生的机制。作为这些研究的补充，将对DNA和马达之间接触的性质和强度及其在机械力化学循环的不同阶段的调节进行表征。最后，我们将确定马达的其他非催化元件的参与，如头尾连接器和通过包装过程中在衣壳内产生的内部DNA压力来调节马达的动力学。为了进行这些研究，我们将利用我们实验室最先进的光学镊子仪器。这台仪器将使我们有可能以前所未有的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射线结晶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，并用于指导这一过程模型的开发。 公共卫生相关性：我们建议使用遗传学、生物化学和生物物理方法研究负责噬菌体29基因组压缩的包装马达的详细分子机制。具体地说，我们将使用单分子光钳方法来表征这个环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，我们希望研究不断增加的DNA内部压力如何调节包装过程中马达的动态。