Search

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.

A variety of replication fork traps have recently been characterised in Enterobacterales, unveiling two different types of architecture. Of these, the degenerate type II fork traps are commonly found in Enterobacteriaceae such as Escherichia coli. The newly characterised type I fork traps are found almost exclusively outside Enterobacteriaceae within Enterobacterales and include several archetypes of possible ancestral architectures. Dickeya paradisiaca harbours a somewhat degenerate type I fork trap with a unique Ter1 adjacent to tus gene on one side of the circular chromosome and three putative Ter2-4 sites on the other side of the fork trap. The two innermost Ter1 and Ter2 sites are only separated by 18 kb, which is the shortest distance between two innermost Ter sites of any chromosomal fork trap identified so far. Of note, the dif site is located between these two sites, coinciding with a sharp GC-skew flip. Here we examined and compared the binding modalities of E. coli and D. paradisiaca Tus proteins for these Ter sites. Surprisingly, while Ter1-3 were functional, no significant Tus binding was observed for Ter4 even in low salt conditions, which is in stark contrast with the significant non-specific protein-DNA interactions that occur with E. coli Tus. Even more surprising was the finding that D. paradisiaca Tus has a relatively moderate binding affinity to double-stranded Ter while retaining an extremely high affinity to Ter-lock sequences. Our data revealed major differences in the salt resistance and stability between the D. paradisiaca and E. coli Tus protein complexes, suggesting that while Tus protein evolution can be quite flexible regarding the initial Ter binding step, it requires a highly stringent purifying selection for its final locked complex formation.

InEscherichia coli, DNA replication termination is orchestrated by two clusters ofTersites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Tercomplex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at someTersites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermostTersites (TerA-EandG) were significantly bound by Tus. We also found that a single ectopic insertion ofTerBin its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’Tersites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture exclusively found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow prototypical Tus-dependent DNA replication fork trap consisting of only twoTersites.Graphical Abstract

Tus is a protein involved in DNA replication termination that binds specific DNA sequences (Ter) located around the terminus region of the chromosome in Enterobacterales. Tus and Ter form a unique monomeric protein-DNA complex which is one of strongest of its kind. A fascinating aspect of Tus-Ter is its ability to dramatically change conformation into a locked structure upon progression of a replication fork towards the non-permissive face of the complex. Over the last two decades, several new technologies have emerged harnessing the unique and interesting properties of this fascinating DNA-binding protein. This review highlights the important properties of the Tus-Ter complex and their exploitation for the development of diverse and novel ultrasensitive detection devices as well as innovative genomic and proteomic platform technologies. A variety of ex vivo and in vivo bioanalytical applications are discussed, including immuno-PCR diagnostic, bioassay and protein array technologies that are broadly relevant to the fields of cancer biology, microbiology and immunology. A perspective on future research and applications is provided.