Figure 1.

Mutational analysis of the Tus-Ter complex when reconstituted in S. cerevisiae. Yeast strains engineered with either restrictive (blocking) or permissive (non-blocking) 3xTerB modules adjacent to ARS305 (on ChrIII) and ARS607 (on ChrVI) were transformed with a low-copy GAL1-regulated plasmid containing HA-tagged wild-type Tus, or HA-Tus with E47Q, E49K, or F140A substitutions. The 3xTerB modules examined here were arranged in either (A) the ChrIII ^RESTRICTIVE / ChrVI ^permissive orientation, or (B) the reciprocal ChrIII ^permissive / ChrVI ^RESTRICTIVE configuration. At both of these genomic loci, replication forks emanating from either ARS305 or ARS607 replicate these 3xTerB modules from left to right. Cultures were synchronized in G1 with α-factor pheromone, and expression of Tus was induced for 2.5 hours during the cell synchronization step. Following a 35 min release from G1-arrest, cells were harvested for 2D gel analysis (left panels), or Western blotting for HA-Tus (right panels). The 2D gel images show DNA replication intermediates detectable at (a BamHI-HindIII fragment of) ChrIII and (a HindIII-HindIII fragment of) ChrVI restriction fragments. Paused RFs at Tus-Ter are indicated by the black arrow. Western blotting confirmed equivalent levels of wild type and mutated HA-Tus proteins at this time point. A control (uninduced HA-Tus) sample was also included as a negative control for the Western blot analysis. Membranes were stained with Ponceau S to confirm equivalent protein loading (lower right panels).