Replication of circular chromosome initiates at a unique sequence termed origin of replication and proceeds bidirectionally until the two replication forks meet in the terminus region located opposite the origin. In this region are located a series of sites, called termination or Ter sites, that block replication forks moving in one direction “non-permissive face” but not the other “permissive face”. In E. coli the Ter sites bind the Tus replication terminator protein, and are arranged to create a replication fork trap that allows forks to enter but not to leave the terminus region. In one model, this polar arrest is induced by the communication between DnaB, the helicase that unwinds the parental DNA strands, and non-permissive Tus/Ter complex. In another model, DNA unwinding induces flipping of a conserved cytosine base in Ter (C6) that create new interactions in a cytosine-binding pocket in Tus and form a stable non-permissive Tus/Ter.

We used single-molecule imaging to monitor the fate of two in vitro reconstituted replication forks, E. coli and bacteriophage T7, as they approach a permissive or non-permissive Tus/Ter complexes. Consistent with the in vivo results, we observe 50% efficiency of E. coli fork stoppage by the non-permissive Tus/Ter. The probability of fork stoppage is entirely rate dependent whereby faster moving forks are arrested less efficiently than slower one. In contrast, the 10-fold slower bacteriophage T7 fork is stopped with 100% efficiency arguing against a specific DnaB and Tus interaction in mediating fork arrest. Using altered Ter sites we demonstrate that C6-base flipping in the Tus/Ter non-permissive complex is the primary mechanism that mediates polar arrest. We also observed a transient pause in DNA synthesis prior to fork stoppage by the Tus/Ter non-persmissive complex. We propose the presence of a secondary signal prior to full stoppage whose mechanism is currently under investigation.