TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

在大肠杆菌染色体的末端区域，DNA 复制分叉被阻止在特定的DNA序列处，称为Ter 网站。DNA复制的抑制是由Tus蛋白介导的，Tus蛋白 以高亲和力与Ter位点结合。Tus与Ter位点的结合 形成不对称的蛋白质-DNA复合体，显示出功能的极性； 也就是说，它会从一个方向停止复制派生，但不会停止 其他的。因此，Tus-Ter复合体形成了一种取向相关的势垒 与DNA复制的进程有关。 在发现土族人和美国人的六年里 在更多的地点，已经取得了重大进展的特点 Tus与其同源结合序列的相互作用及Tus之间的相互作用 以及复制停止期间的复制体。尽管如此，分子 TUS介导的DNA复制抑制机制尚不清楚。 同样，我们对TUS蛋白结构域的了解 参与DNA结合或阻止DNA复制的功能是 基本上是不完整的。这项提案中概述的实验包括 旨在更好地理解结构-功能 Tus蛋白与复制抑制机制的关系。 我们提出三个具体目标： 1)分离复制抑制基因外抑制物 基因技术用于识别参与基因产物 复制停止过程。 2)确定准确测量TUS功能和 利用此系统检查有关流程的基本问题并 复制抑制机制。 3)研究TUS蛋白的功能结构域 以下办法：(A)Tus与Ter网站的交叉链接，(B) 使蛋白质单独结晶或作为蛋白质-DNA复合体结晶，以及 (C)连接扫描仪对蛋白质的各个结构域进行突变。

In the terminus region of the Escherichia coli chromosome, DNA replication forks are arrested at specific DNA sequences, called Ter sites. Arrest of DNA replication is mediated by the Tus protein, which binds to the Ter sites with high affinity. Binding of Tus to a Ter site forms an asymmetric protein-DNA complex that shows polarity of function; that is, it halts replication forks from one direction, but not the other. Thus, the Tus-Ter complex forms an orientation-dependent barrier to the progression of DNA replication. In the six years that have passed since the identification of Tus and the Ter sites, significant advances have been made characterizing the interactions between Tus and its cognate binding sequence and between Tus and the replisome during replication arrest. Nonetheless, the molecular mechanism of Tus-mediated arrest of DNA replication remains unknown. Likewise, our knowledge of the domains of the Tus protein that participate in DNA-binding or the DNA replication arrest function is largely incomplete. The experiments outlined in this proposal are directed towards a greater understanding of structure-function relationships in the Tus protein and the mechanism of replication arrest. We propose three specific goals: 1) Isolate extragenic suppressors of replication arrest using standard genetic techniques to identify gene products that participate in the replication arrest process. 2) Identify an in vitro system that accurately measures Tus function and utilize this system to examine basic questions about the process and mechanism of replication arrest. 3) Investigate the functional domains of the Tus protein using the following approaches: (a) cross-linking of Tus to a Ter site, (b) crystallizing the protein either alone or as a protein-DNA complex, and (c) linker scanner mutagenesis of individual domains of the protein.