MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

T4 DNA 复制复合体中的分子相互作用

• 批准号: 2608783
• 负责人: PETER H VON HIPPEL
• 金额: $ 23.46万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608783
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; X ray crystallography; adenosinetriphosphatase; atomic force microscopy; bacteriophage T4; bioenergetics; chemical kinetics; cryoelectron microscopy; crystallization; enzyme complex; enzyme mechanism; fluorescence spectrometry; helicase; intermolecular interaction; laboratory mouse; laboratory rabbit; protein structure function; thermodynamics; virus DNA; virus genetics; virus protein

The next stages of a continuing physical biochemical study of the structural and functional interactions of the protein and nucleic acid components of the bacteriophage T4 DNA replication system are outlined in this proposal. We will continue to study the mechanism of action of each functional subset of proteins of this in vitro replication system, focussing on how these subsets are assembled into the integrated DNA replication complex. These subsets include the central DNA polymerase (gene 43 protein), the polymerase accessory proteins complex (genes 44/62 and 45 proteins), and the helicase -primase complex ((genes 41 and 61 proteins), each of which also interacts with the T4 single-stranded DNA binding (gene 32) protein. During the next granting period we will pursue, in particular, the following specific aims. (i) We will continue our physical biochemical studies of the structure and function of the five-protein polymerase holoenzyme complex, focussing primarily on how the processivity ring (gp45) is loaded on to the primer-template junctions within the replication fork in a PuTP-driven "loading' reaction carried out by the gp44/62 sub-assembly of the polymerase accessory proteins, and how encounter with Okazaki fragments in lagging strand synthesis might trigger rapid release of the processivity ring, and thus the holoenzyme from the template; (ii) to elucidate the mechanisms whereby the gp41 hexamer functions as a helicase of double-stranded DNA (and as a unidirectional "translocase" on single- stranded DNA); (iii) to characterize the interactions between the helicase (gp41) and the primase (g61) components of the functional primosome of the T4 DNA replication, system, and to determine how these interactions modulate and regulate the helicase (and translocase) activities of gp41, as well as control the primary activity of gp61; (iv) to use low resolution (but "real time") structural information obtained by cryoelectron microscopy, and detailed molecular structural information obtained by x-ray crystallography to learn how these individual proteins and protein subassemblies operate and interact as molecules in discharging their physiological roles; and (v) to use the knowledge gathered in these studies to examine the integration of these subassemblies into a functional seven- protein (or eight-protein, if the helicase loading factor gp59 is also required) complex that is capable of elongating DNA at the physiological rate and with physiological processivity and fidelity. These studies now appear to be particularly directly health related, since considerable recent work has shown that both the processivity and the helicase components of the T4 system may be very analogous to the eukaryotic proteins that are involved in connecting DNA replication and repair to cell cycle control and tumorogenesis.

下一个阶段的持续物理生化研究， 蛋白质和核酸的结构和功能相互作用 噬菌体T4 DNA复制系统的组成部分概述于 这个提议。 我们将继续研究每种药物的作用机制。 该体外复制系统的蛋白质的功能子集， 重点是这些亚群如何组装成整合的DNA 复制复合体 这些子集包括中央DNA聚合酶 (gene 43蛋白），聚合酶辅助蛋白复合物（基因44/62 和45蛋白质），以及解旋酶-引发酶复合物（（基因41和61 蛋白质），其中每一个也与T4单链DNA相互作用 结合（基因32）蛋白。 在下一个资助期内，我们会特别就 具体目标。 (i)我们将继续我们的物理生化 五蛋白聚合酶的结构和功能研究 全酶复合体，主要关注持续合成能力环（gp 45） 被加载到复制叉内的引物-模板连接处， 在由gp 44/62子组件进行的PuTP驱动的“加载”反应中， 的聚合酶辅助蛋白，以及如何遇到冈崎 滞后链合成中的片段可能会触发 合成环，并因此从模板全酶;（ii） 阐明gp 41六聚体作为解旋酶发挥作用的机制 双链DNA（以及作为单链DNA上的单向“易位酶”， （iii）表征解旋酶之间的相互作用 （gp 41）和引发酶（g61）的功能性引发体的组分。 T4 DNA复制，系统，并确定这些相互作用 调节和调节gp 41的解旋酶（和转位酶）活性， 以及控制gp 61的主要活性;（iv）使用低分辨率 (but“真实的时间”）结构信息 显微镜和详细的分子结构信息获得的x射线 晶体学来了解这些单个蛋白质和蛋白质 电子能像分子一样运作和相互作用， 生理作用;以及（v）利用这些研究中收集的知识 将这些功能整合到一个功能性的七个功能中， 蛋白质（或八蛋白质，如果解旋酶加载因子gp 59也是 所需的）复合物，其能够在生理条件下延伸DNA。 速率和生理持续合成能力和保真度。 这些研究现在 似乎与健康直接相关，因为 最近的研究表明，合成能力和解旋酶 T4系统的组成部分可能非常类似于真核生物。 参与连接DNA复制和修复细胞的蛋白质 周期控制和肿瘤发生。