Mutations in the Escherichia coli Tus Protein Define a Domain Positioned Close to the DNA in the Tus-Ter Complex

Aikaterini Skokotas,Hiroshi Hiasa,Kenneth J Marians,Leslie O'Donnell,Thomas M Hill

doi:10.1074/jbc.270.52.30941

Abstract

A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication. We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y. TusP42S and TusE49K arrest DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency. The loss of replication arrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins. TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1/2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest. When tested in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo and in vitro assays. Finally, the half-lives of the mutant protein-DNA complexes suggested that the domain of Tus where these mutations are located is positioned close to the DNA in the Tus-Ter complex.

Highlights

A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication
DNA replication forks traversing the chromosome of Escherichia coli are halted at specific protein-DNA complexes composed of the Tus protein bound to a Ter site (for reviews, see Hill (1992), Yoshikawa and Wake (1993), and Baker (1995))
The Ter sites that have been identified in the E. coli chromosome (TerA-TerF) are oriented to permit DNA replication in the origin to terminus direction, but block DNA replication forks moving in the terminus to origin direction

Summary

Introduction

A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication. TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1⁄2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest. On the long substrate where true helicase unwinding is occurring, Tus was not able to block DnaB progression, suggesting that binding of Tus to Ter alone does not inhibit DnaB and that protein-protein interactions must be involved. This observation is supported by the recently characterized mutant TusA173V (Skokotas et al, 1994). This result indicated that a large decrease in the affinity of TusA173V for TerB did not lead to a corresponding decrease in replication arrest activity, as would be expected if binding alone was responsible for the inhibition of DNA replication

Methods

Results

Conclusion