Site-Directed Mutagenesis and Yeast Reverse 2-Hybrid-Guided Selections to Investigate the Mechanism of Replication Termination

Abstract

DNA replication in prokaryotes, in budding yeast and in mammalian DNA viruses initiates from fixed origins (ori) and the replication forks are extended in either a bidirectional mode or in some cases unidirectionally (Cvetic and Walter, 2005; Sernova and Gelfand, 2008; Wang and Sugden, 2005; Weinreich et al., 2004). In higher eukaryotes there are preferred sequences located in AT-rich islands that serve as origins (Bell and Dutta, 2002). In many prokaryotes, the two replication forks initiated at ori on a circular chromosome meet each other at specific sequences called replication termini or Ter (Bastia and Mohanty, 1996; Kaplan and Bastia, 2009). The Ter sites bind to sequence-specific DNA binding proteins called replication terminator proteins that allow forks approaching from one direction to be impeded at the terminus, whereas forks coming from the opposite direction pass through the site unimpeded (Bastia and Mohanty, 1996, 2006; Kaplan and Bastia, 2009). Therefore, the mode of fork arrest is polar. The polarity of fork arrest in Escherichia coli and Bacillus subtilis is caused by the complexes of the terminator proteins called Tus and RTP (Replication Terminator Protein), respectively, with the cognate Ter sites to arrest the replicative helicase (such as DnaB in case of E. coli) in a polar mode (Kaul et al., 1994; Khatri et al., 1989; Lee et al., 1989; Sahoo et al., 1995). What is the mechanism of polar fork arrest and what might be the physiological functions of Ter sites? Using E. coli as the main example, with the aid of the techniques of site-directed mutagenesis, yeast reverse 2-hybrid based selection of random mutations (described below), and biochemical characterizations of the mutant forms of the Tus protein, many aspects of the mechanism of replication fork arrest at Tus-Ter complexes have been determined. This and a brief description of the current state of the knowledge of replication termination in eukaryotes have also been reviewed below.