TERMINATION OF DNA REPLICATION IN ESCHERICHIA COLI

在大肠杆菌染色体的末端区域，DNA 复制叉在称为Ter的特定DNA序列处被捕获 网站. Tus蛋白介导DNA复制的停滞， 以高亲和力结合Ter位点。 Tus与Ter位点的结合 形成显示功能极性的不对称蛋白质-DNA复合物; 也就是说，它从一个方向停止复制分叉，但不从 其他.因此，Tus-Ter复合物形成了依赖于取向的势垒 DNA复制的进展。 在查明图斯和 在这些地点，已经取得了重大进展， Tus与其同源结合序列之间以及Tus与其同源结合序列之间的相互作用 和复制体之间的关系。尽管如此， Tus介导的DNA复制停滞的机制仍然未知。 同样，我们对Tus蛋白结构域的了解， 参与DNA结合或DNA复制阻滞功能的是 基本上不完整。该提案中概述的实验是 旨在更好地理解结构-功能 Tus蛋白与复制停滞机制的关系。 我们提出三个具体目标： 1)使用标准品分离复制停滞的基因外抑制因子 基因技术，以确定基因产品，参与 复制抑制过程。 2)确定一种准确测量Tus功能的体外系统， 利用这个系统来检查有关过程的基本问题， 复制停滞机制。 3)研究Tus蛋白的功能结构域， 以下方法：（a）Tus与Ter位点的交联，（B） 使所述蛋白质单独结晶或作为蛋白质-DNA复合物结晶，和 (c)连接子扫描仪诱变蛋白质的各个结构域。

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.