ISOLATION AND CHARACTERIZATION OF MUTANTS OF TUS, THE REPLICATION ARREST PROTEIN OF ESCHERICHIA-COLI

Mutations in the tus gene of Escherichia coil, which encodes the repli cation arrest protein Tus, were isolated using a selection scheme base d on the plasmid pHV750T2(+), which transforms tus mutants at a much h igher frequency than wild type strains. Seven mutants containing singl e nucleotide substitutions were isolated, and all of these mutants sho wed reduced or complete loss of DNA binding and replication arrest act ivity. Two of the mutant proteins, containing a valine (A173V) or thre onine (A173T) in place of the alanine normally found at amino acid 173 , were purified and characterized further. A173T had a 4100-fold lower affinity for Ter sites than wild type Tus and was unable to halt DNA replication in vivo or inhibit DnaB-catalyzed strand displacement in a n in vitro helicase assay. A173V showed a 130-fold lower affinity for Ter sites than wild type Tus but was still able to arrest DNA replicat ion in vivo, suggesting that protein-protein interactions were respons ible for Tus-mediated arrest of DNA replication. In addition, we found that A173V was a weak inhibitor of DnaB-catalyzed strand displacement in vitro, yet halted DNA replication in vivo at 75% of the efficiency of wild type Tus. We concluded from these observations that the stand ard in vitro helicase assay was inadequate for measuring Tus activity.