Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

Abstract

The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

Keywords: DnaB helicase; MCM helicase; RecQ helicase; homologous recombination repair; replication fork.

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).