Arrest of DNA Replication in E. coli

大肠杆菌中的 DNA 复制总是从环状染色体上的特定点开始，称为复制起点。 从起点开始的两个复制叉沿着染色体的每一半行进，并在距离起点 180° 的一个称为终点的区域相遇。 在染色体的末端区域，复制叉被阻止在称为 Ter 位点的特定 DNA 序列处。 DNA 复制的抑制是由 Tus 蛋白介导的，该蛋白与 Ter 位点结合形成不对称的蛋白-DNA 复合物。 Tus-Ter 复合物显示出功能的极性；也就是说，它会阻止复制叉从一个方向接近，但不会阻止从另一个方向接近。 因此，Tus-Ter 复合物构成了 DNA 复制进程的方向依赖性障碍。 该项目的主要目标是了解启迪阻止复制机制的机制。 该实验室的初步实验表明，位于大肠杆菌拓扑异构酶 I (topA) 基因附近或基因中的突变会抑制野生型 Tus-Ter 复合物的复制停滞。 生物化学、遗传和分子方法将用于表征这些突变并确定绕过 Tus-Ter 复合物的机制。 该实验室还发现 Tus 中某些氨基酸的突变会损害复制停滞活性，但不会显着损害 DNA 结合。 为了进一步扩展这些研究，将使用随机诱变来鉴定有助于其功能的 Tus 结构域，然后进行针对特定氨基酸的定点诱变。 然后将使用体内和体外测定来评估突变型 Tus 蛋白阻止 DNA 复制的能力。 从这项研究中获得的信息将有助于阐明 Tus 停止 DNA 复制的机制，并阐明复制抑制系统在细菌中发挥的生理作用。 此外，了解大肠杆菌中的 Tus-Ter 系统将增加我们对酵母和高等真核生物的了解，这些生物也具有复制抑制系统。

DNA replication in Escherichia coli is always initiated from a specific point on the circular chromosome, called the origin of replication. The two replication forks initiated from the origin travel along each half of the chromosome and meet 180 from the origin in a region called the terminus. In the terminus region of the chromosome, 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 to form an asymmetric protein-DNA complex. The Tus-Ter complex shows polarity of function; that is, it halts replication forks approaching from one direction but not the other. Thus, the Tus-Ter complex constitutes an orientation-dependent barrier to the progression of DNA replication. The primary objective of this project is to understand the mechanism by which Tus arrests the replication machinery. Preliminary experiments from this lab have suggested that mutations mapping near to or in the gene for topoisomerase I (topA) of E. coli suppress replication arrest by a wild-type Tus-Ter complex. Biochemical, genetic, and molecular approaches will be used to characterize these mutations and to identify the mechanism by which the Tus-Ter complexes are bypassed. This lab has also found that mutations at certain amino acids in Tus impair replication arrest activity without significantly impairing DNA binding. To extend these studies further, random mutagenesis will be used to identify the domains of Tus that contribute to its function, followed by site-directed mutagenesis to target specific amino acids. The ability of the mutant Tus proteins to arrest DNA replication will then be assessed using in vivo and in vitro assays. The information gained from this research will help elucidate the mechanism by which DNA replication is halted by Tus and shed light on the physiological role that replication arrest systems play in bacteria. In addition, understanding the Tus-Ter system in E. coli will increase our understanding of yeast and higher eukaryotes, which also have replication arrest systems.