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.
