A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli

Abstract

During chromosome synthesis in Escherichia coli, replication forks are blocked by Tus bound Ter sites on approach from one direction but not the other. To study the basis of this polarity, we measured the rates of dissociation of Tus from forked TerB oligonucleotides, such as would be produced by the replicative DnaB helicase at both the fork-blocking (nonpermissive) and permissive ends of the Ter site. Strand separation of a few nucleotides at the permissive end was sufficient to force rapid dissociation of Tus to allow fork progression. In contrast, strand separation extending to and including the strictly conserved G-C(6) base pair at the nonpermissive end led to formation of a stable locked complex. Lock formation specifically requires the cytosine residue, C(6). The crystal structure of the locked complex showed that C(6) moves 14 A from its normal position to bind in a cytosine-specific pocket on the surface of Tus.

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

“…Tus F140A fails to support an efficient "lock" mechanism when the Ter site is partially single stranded at the non-permissive end, because it cannot accommodate the base-flipped Ter-C6 residue. 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.…”

“…By placing the 6xTer array within a reporter of mammalian HR, targeted to a specific locus of mouse embryonic stem (ES) cells, we found that transient expression of wild type Tus triggers HR at the 6xTer array, while expression of a mutant form of Tus (H144A) that has a low affinity for Ter failed to induce HR in the same cells. 14,15 Several lines of evidence indicate that Tus/Ter-induced HR in mammalian cells entails the processing of 2 converging forks arrested at the Tus/Ter fork block. Tus/Ter-induced HR predominantly generates "short tract" gene conversions (STGC)-a conservative HR outcome.…”

“…7 The hereditary breast/ovarian cancer predisposition genes, BRCA1 and BRCA2, in concert with other Fanconi anemia genes and other HR genes, have been implicated in regulating HR/SCR in response to replication fork stalling, underscoring the importance of this process for cancer predisposition. [8][9][10] We recently adapted the Escherichia coli Tus/Ter replication fork arrest complex [11][12][13][14] to provoke site-specific replication fork stalling and chromosomal HR in mammalian cells. 15 In E. coli, binding of the monomeric Tus protein to the asymmetrical »23 bp Ter site provokes polar replication fork arrest, depending on whether the replicative helicase DnaB arrives at the non-permissive or permissive end of Ter.…”

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

“…NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4574 ARTICLE characterized C-T substitution 22 in each of the TerB sites adjacent to ARS305 abolished RF pausing ( Supplementary Fig. 2).…”

“…Two models have been proposed to explain how Tus-Ter functions as a polar RF barrier in E. coli. The 'mousetrap' model of Tus-Ter RF pausing posits that a specific interaction is required between Tus and a critical 'locking cytosine' residue within TerB that is revealed by DNA strand separation 22 . An alternative model proposes that RF arrest at Tus-Ter is mediated by specific protein-protein interactions in E. coli 23,24 .…”