Delineation of the Ancestral Tus-Dependent Replication Fork Trap

Abstract

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus–Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA–E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

Interplay between chromosomal architecture and termination of DNA replication in bacteria

“…In a recent study by the Schaeffer lab, ChiP-Seq was used to identify which ter sites are actually occupied by Tus protein. The authors identified ter sites A–E and G ( Figure 1C ), whereas no Tus occupancy was observed at any of the other ter sites ( Toft et al, 2021 ).…”

“…The highly conserved ter core (6, 8–18) is highlighted by a darker grey. Tus binding ( Toft et al, 2021 ), in vivo fork blocking observed by 2D gel electrophoresis ( Duggin and Bell, 2009 ) and analysis whether a ter site is part of an open reading frame ( Duggin and Bell, 2009 ; Goodall et al, 2021 ) are indicated (see text for further details).…”

“…The precise molecular mechanism for this regular progression of synthesis beyond ter sites C and B is not clear. However, the ChIP-Seq data from the Schaeffer lab would predict that escape synthesis should proceed past terF before getting blocked by terG ( Toft et al, 2021 ). This is indeed precisely what we observed, supporting the idea that terF is not blocking the progression of forks in normal cells, whereas terG can block ongoing synthesis in wild type cells ( Figure 2C ).…”

“…A prediction from this hypothesis would be that there might be chromosomes with a single ter site limiting progression of one of the replichores, and where the tus gene is associated with this innermost ter site. In a recent study by Toft and colleagues it was indeed highlighted that two distinct fork trap architectures exist ( Toft et al, 2021 ). The fork trap system in E. coli is classed as a type II system, because the ter site regulating transcription of the tus gene is terB , which is a secondary rather than the innermost ter site ( Toft et al, 2021 ).…”

“…In a recent study by Toft and colleagues it was indeed highlighted that two distinct fork trap architectures exist ( Toft et al, 2021 ). The fork trap system in E. coli is classed as a type II system, because the ter site regulating transcription of the tus gene is terB , which is a secondary rather than the innermost ter site ( Toft et al, 2021 ). Outside of the Enterobacteriaceae, another system exists which the authors classified as a type I system.…”