MUTATIONS IN THE ESCHERICHIA-COLI TUS PROTEIN DEFINE A DOMAIN POSITIONED CLOSE TO THE DNA IN THE TUS-TER COMPLEX

A new genetic screen for mutations in the tus gene of Escherichia coil has been devised that selects for Tus proteins with altered ability t o arrest DNA replication. We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y, TusP42S and TusE49K arre st DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency. The loss of replication a rrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins, TusE49K formed a mor e stable protein-DNA complex than wild-type Tus (t(1/2) 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 te sted in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo an d in vitro assays. Finally, the half-lives of the mutant protein-DNA c omplexes suggested that the domain of Tus where these mutations are lo cated is positioned close to the DNA in the Tus-Ter complex.