Mechanisms of Viral DNA Packaging

描述（由申请人提供）：在许多病毒中，通过ATP依赖性门脉马达的作用，一个空的“前体”被组装并随后充满DNA。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靶点。在这个应用中，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对噬菌体F29门脉马达包装过程的单分子研究。这个噬菌体是一个理想的系统来研究这一过程作为一个强大的体外包装试验已经可用。在封装过程中，DNA被压缩到接近结晶的密度，克服了由于静电排斥、DNA弯曲刚度和熵引起的能量惩罚。由于该马达由ASCE atp酶的五聚体环组成，其研究将为该家族的其他成员以及更大的AAA+环atp酶超家族的运作提供重要的启发，这些超家族负责大量的细胞功能，从蛋白质的展开和降解到原核生物的染色体分离。我们建议在每个ATP酶的操作周期中详细描述各种化学和机械事件（即ATP结合，水解，产物释放，易位等），并建立环中单个亚基周期之间的精确时间或协调。我们还将建立电机产生扭矩的能力，其大小和相对于产生线性力的产生机制。这些研究将由DNA和马达之间接触的性质和强度的特征及其在机械化学循环的各个阶段的调制来补充。最后，我们将建立电机的其他非催化元件的参与，如头尾连接器，以及在封装过程中由衣壳内部产生的内部DNA压力对电机动力学的调节。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。生物物理测量结果将与x射线晶体学和低温电子显微镜（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。

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.