Organization of Bacteriophage T4 DNA Replication Complex

可视化噬菌体组分的初步实验 在酶活性条件下，通过冷冻EM检测T4复制装置 条件下，成功识别出独特的组件 复制辅助蛋白与DNA结合的复合物。 这些组件需要连续的ATP水解，具有短的 寿命，似乎只包括少数的蛋白质 在样品中;因此，它们很难分离， 用冷冻电镜以外的方法进行分析。 下一步 项目是确定的组成和安排， 这些复合物中的蛋白质，通过冷冻EM，位点- 特异性标记（亚基特异性抗体和共价标记） 标签）和生物化学分析。 形成机理 和易位的这些结构将被检查， 尝试确定与T4 DNA聚合酶的相互作用 了解辅助蛋白在这个过程中的作用 复制。 T4复制的其他元素 机器，DNA解旋酶和RNA引物酶，也将被 通过这些方法进行检查，以确定其在 酶活性复合物及其结构关系 到聚合酶。 %%% 基因组的准确和有效复制是一个关键 所有生命的过程。 取得了很大进展，主要是 生化分析，在阐明分子机制， DNA复制。 所涉及的酶的复杂性 比最初预期的要大。 的相互作用 DNA复制所需的许多蛋白质中，有一种已被证明是 特别难以直接分析，部分原因是 活性复制复合物的瞬时性质。 新 电子显微镜技术，特别是 用低温方法进行最小扰动的标本保存 （cryo-EM），开辟了新的途径，以接近研究 活性的、化学敏感的大分子组装体， 催化DNA复制的酶 生化检查 许多不同生物体的复制机制已经确定 该过程的共同要素，允许结构 获得了关于简单系统（噬菌体和细菌）的信息， 与高等生物的基因组复制有关。 工作 这里提出的是一种尝试，以进一步了解 DNA复制机制的结构功能研究 噬菌体T4蛋白质的相关复合物 复制装置（具有DNA模板和产物），以及 将结构分析与酶促反应中的步骤关联起来 活动

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.