Arrest of DNA Replication in E. coli

大肠杆菌中 DNA 复制的抑制

• 批准号: 9816998
• 负责人: Thomas Hill
• 金额: $ 30万
• 依托单位: University of North Dakota Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9816998&HistoricalAwards=false
• 关键词: Arrest; DNA; Replication; E.; coli

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.

大肠杆菌中的DNA复制总是从圆形染色体上的一个特定点开始，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移动，并在原点180处的一个称为末端的区域相遇。在染色体的末端区域，复制叉被固定在特定的DNA序列上，称为Ter位点。阻止DNA复制是由Tus蛋白介导的，它与Ter位点结合形成不对称的蛋白质-DNA复合物。Tus-Ter复合物具有功能极性；也就是说，它阻止复制分叉从一个方向靠近，而不是从另一个方向靠近。因此，Tus-Ter复合体构成了DNA复制进展的定向依赖屏障。该项目的主要目标是了解Tus阻止复制机制的机制。该实验室的初步实验表明，大肠杆菌拓扑异构酶I （topA）基因附近或内部的突变抑制了野生型Tus-Ter复合物的复制阻滞。生物化学、遗传和分子方法将被用来描述这些突变，并确定绕过Tus-Ter复合物的机制。该实验室还发现，在某些氨基酸的突变损害复制阻滞活性，但不显着损害DNA结合。为了进一步扩展这些研究，将使用随机诱变来识别有助于其功能的Tus结构域，然后使用定点诱变来靶向特定氨基酸。突变的Tus蛋白阻止DNA复制的能力将在体内和体外进行评估。从这项研究中获得的信息将有助于阐明DNA复制被Tus阻止的机制，并阐明复制阻止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级真核生物的了解，它们也有复制阻止系统。