Organization of Bacteriophage T4 DNA Replication Complex

在酶活性条件下，通过冷冻-EM显示噬菌体T4复制装置的组件的初步实验，成功地鉴定了与DNA结合的复制辅助蛋白复合体的独特组装。这些组件需要持续的ATP水解，寿命短，似乎只占样本中蛋白质的一小部分；因此，很难通过冷冻-EM以外的方法分离它们进行结构分析。该项目的下一步是通过结合冷冻-EM、位点特异性标记(亚基特异性抗体和共价标记)和生化分析来确定这些复合体中蛋白质的组成和排列。将研究这些结构的形成和移位的机制，并确定它们与T4DNA聚合酶的相互作用，以试图了解辅助蛋白在复制过程中的作用。T4复制机制的其他元素，DNA解旋酶和RNA引物酶，也将通过这些方法进行检查，以确定它们在具有酶活性的复合体中的排列以及它们与聚合酶的结构关系。基因组的准确和有效复制是生命中的一个关键过程。在阐明DNA复制的分子机制方面，主要是通过生化分析，已经取得了很大的进展。所涉及的酶的复杂性比最初预期的要大。DNA复制所需的许多蛋白质之间的相互作用已被证明特别难以直接分析，部分原因是活性复制复合体的瞬变性质。电子显微镜中的新技术，特别是使用最小扰动低温保存方法(低温-EM)，为研究活性的、化学敏感的大分子组件开辟了新的途径，例如那些催化DNA复制的组件。对许多不同生物体中复制机制的生化检查已经确定了该过程的共同元素，这使得获得的关于简单系统(噬菌体和细菌)的结构信息与高等生物体中的基因组复制有关。本工作旨在通过对T4噬菌体复制装置蛋白质(与DNA模板和产物)的功能相关复合体的结构研究来加深对DNA复制机制的理解，并将结构分析与酶活性的步骤相关联。

Initial experiments to visualize components of the bacteriophage T4 replication apparatus by cryo-EM, under enzymatically active conditions, have successfully identified distinctive assemblies of a complex of the replication accessory proteins bound to DNA. These assemblies require continual ATP hydrolysis, have short lifetimes, and appear to comprise only a minority of the protein in the sample; hence they are difficult to isolate for structural analysis by means other than cryo-EM. The next step in this project is determining the composition and arrangement of the proteins in these complexes, by a combination of cryo-EM, site- specific labelling (subunit-specific antibodies and covalent labels), and biochemical analysis. The mechanism of formation and translocation of these structures will be examined, and their interaction with the T4 DNA polymerase will be determined, to try to understand the role of the accessory proteins in the process of replication. The other elements of the T4 replication machinery, the DNA helicase and RNA primase, will also be examined by these methods, to determine their arrangement in enzymatically active complexes and their structural relationship to the polymerase. %%% Accurate and efficient replication of the genome is a critical process in all life. Much progress has been made, primarily by biochemical analysis, in elucidating the molecular mechanism of DNA replication. The complexity of the enzymes involved is greater than had been initially anticipated. The interactions among the many proteins required for DNA replication have proven particularly difficult to analyze directly, in part due to the transient nature of active replication complexes. New technologies in electron microscopy, in particular the use of minimally-perturbing specimen preservation by cryogenic methods (cryo-EM), has opened new avenues to approach the study of active, chemically-sensitive macromolecular assemblies such as those which catalyze DNA replication. Biochemical examination of replication mechanisms in many different organisms has identified common elements of the process, which allow structural information gained about simple systems (phage and bacteria) to be related to genome replication in higher organisms. The work proposed here is an attempt to further the understanding of the mechanism of DNA replication by structural study of functionally relevant complexes of the proteins of the bacteriophage T4 replication apparatus (with DNA templates and products), and to correlate the structural analysis to steps in the enzymatic activity.