The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

Abstract

Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.

Spatial separation of replisome arrest sites influences homologous recombination quality at a Tus/Ter-mediated replication fork barrier

“…14 However, Tus F140A exhibits a higher affinity than wtTus for duplex Ter 14 Larsen et al found that wtTus mediates polar replication fork arrest at a Ter array in S. cerevisiae. 22 The same authors found that Tus F140A is unable to generate an efficient replication fork barrier when bound to a chromosomal Ter array in S. cerevisiae, even when the majority of replication forks arrive at the non-permissive face of Tus/Ter. 21 In contrast, Tus/Ter-mediated replication fork arrest and ensuing HR in mammalian cells is predominantly non-polar and does not require the C6 lock mechanism.…”

“…In contrast, Larsen et al found that Tus/Ter mediates polar replication fork arrest in Saccharomyces cerevisiae. 21,22 Indeed, Tus F140A is defective for replication fork arrest at a Ter array in S. cerevisiae. Thus, current evidence suggests that Tus/Ter mediates replication fork arrest through distinct mechanisms in yeast and mammalian cells.…”

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

“…8 Three groups have independently demonstrated recently that Tus-Ter can function as an RF barrier when reconstituted in yeast, 9 mouse, 10 or human cells (S. N. Powell, unpublished observation). The heterologous nature of Tus-Ter when introduced into non-bacterial organisms is an important aspect, because endogenous (programmed) RF pauses are established and dealt with differently than are 'alien' RF impediments.…”

“…The heterologous nature of Tus-Ter when introduced into non-bacterial organisms is an important aspect, because endogenous (programmed) RF pauses are established and dealt with differently than are 'alien' RF impediments. 9,11 The Tus-Ter system therefore most likely induces cellular responses similar to those that occur when the replisome encounters certain types of DNA damage. 9,10 Reconstitution of the Tus-Ter system in eukaryotes has already been used successfully to glean important information about the molecular events occurring at stalled RFs.…”

“…9,11 The Tus-Ter system therefore most likely induces cellular responses similar to those that occur when the replisome encounters certain types of DNA damage. 9,10 Reconstitution of the Tus-Ter system in eukaryotes has already been used successfully to glean important information about the molecular events occurring at stalled RFs. Willis et al integrated 6xTer sequences and an associated homologous Keywords: DnaB helicase, replication fork, homologous recombination repair, RecQ helicase, MCM helicase recombination (HR) reporter into the genome of mouse cells to provide direct evidence that Brca1, Brca2 and RAD51 regulate HR at stalled RFs.…”