Arrest of DNA Replication in E. coli

在大肠杆菌中，DNA复制总是从环形染色体上的一个特定的点开始，称为复制起点。从原点开始的两个复制叉沿染色体的每一半移动，并在称为末端的区域从原点相遇180。在染色体的末端区域，复制叉被阻止在特定的DNA序列上，称为Ter位点。DNA复制的抑制是由Tus蛋白介导的，它与Ter位点结合形成不对称的蛋白质-DNA复合体。Tus-Ter复合体显示了功能的两极；也就是说，它阻止了从一个方向接近的复制叉子，但不是另一个方向。因此，TUS-Ter复合体对DNA复制的进行构成了一个方向依赖的障碍。这个项目的主要目标是了解TUS阻止复制机制的机制。这个实验室的初步实验表明，映射到大肠杆菌拓扑异构酶I(TopA)基因附近或基因中的突变可以抑制野生型Tus-Ter复合体的复制停滞。将使用生化、遗传和分子方法来表征这些突变，并确定绕过Tus-Ter复合体的机制。该实验室还发现，Tus中某些氨基酸的突变会削弱复制抑制活性，而不会显著损害DNA结合。为了进一步扩展这些研究，将使用随机突变来确定与其功能有关的Tus结构域，然后使用定点突变来针对特定的氨基酸。然后将使用体内和体外测试来评估突变的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阻止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理解，这些生物也有复制抑制系统。

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.