MOLECULAR INTERACTION IN T4 DNA REPLICATION COMPLEX

继续进行的物理生化研究的下一阶段 蛋白质和核酸的结构和功能相互作用 噬菌体T4 DNA复制系统的组件在 这项提议。我们将继续研究各自的作用机制 这个体外复制系统的蛋白质功能子集， 关注这些子集是如何组装成整合的DNA的 复制复合体。这些亚群包括中心DNA聚合酶 聚合酶辅助蛋白复合体(基因44/62) 和45个蛋白质)，以及解旋酶-底物酶复合体(基因41和61 蛋白质)，每个蛋白质也与T4单链DNA相互作用 结合蛋白(基因32)。 在下一个批准期内，我们会特别推行 遵循特定的目标。(一)我们将继续我们的身体生化 五蛋白聚合酶的结构和功能研究 全酶复合体，主要关注加工性环(Gp45) 被加载到复制分叉内的启动子-模板连接上 在由gp44/62亚组件进行的PuTP驱动的“加载”反应中 聚合酶辅助蛋白，以及如何与冈崎相遇 滞后的链合成中的片段可能会引发快速释放 加工环，从而来自模板的全酶；(Ii)至 阐明gp41六聚体作为解旋酶的作用机制 双链DNA(并作为单链DNA上的单向“转位酶”) 链状DNA)；(Iii)表征解旋酶之间的相互作用 (Gp41)和原始酶(G61)组分。 T4 DNA复制，系统，并确定这些相互作用如何 调控gp41的解旋酶(和转位酶)活性， 以及控制gp61的初级活性；(Iv)使用低分辨率 (但“实时”)通过低温电子获得的结构信息 显微镜和通过x射线获得的详细的分子结构信息 结晶学来了解这些单独的蛋白质和蛋白质 亚组分作为分子运行并相互作用，以释放其 生理作用；以及(V)使用在这些研究中收集的知识 为了研究这些子组件整合成一个功能上的七个- 蛋白质(或八蛋白质，如果解旋酶负载因子GP59也是 必需)能够在生理状态下拉长DNA的复合体 速度，具有生理过程和保真度。现在的这些研究 似乎与健康特别直接相关，因为相当大的 最近的研究表明，加工性和解旋酶 T4系统的组成可能与真核生物非常相似 参与连接DNA复制和修复与细胞的蛋白质 周期控制与肿瘤发生。

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.