A role for 3’ exonucleases at the final stages of chromosome duplication in Escherichia coli

Chromosome duplication initiates via the assembly of replication fork complexes at defined origins, from where they proceed in opposite directions until they fuse with a converging fork. Recent work highlights that the completion of DNA replication is highly complex in both pro- and eukaryotic cells. In this study we have investigated how 3’ and 5’ exonucleases contribute towards the successful termination of chromosome duplication in Escherichia coli. We show that the absence of 3’ exonucleases can trigger levels of over-replication in the termination area robust enough to allow successful chromosome duplication in the absence of oriC firing. Over-replication is completely abolished if replication fork complexes are prevented from fusing by chromosome linearization. Our data strongly support the idea that 3’ flaps are generated as replication fork complexes fuse. In the absence of 3’ exonucleases, such as ExoI, these 3’ flaps can be converted into 5’ flaps, which are degraded by 5’ exonucleases, such as ExoVII and RecJ. Our data support the idea that multiple protein activities are required to process fork fusion intermediates. They highlight the complexity of fork fusions and further support the idea that the termination area evolved to contain fork fusion-mediated pathologies

Too much of a good thing: how ectopic DNA replication affects bacterial replication dynamics

Copyright © 2020 Syeda, Dimude, Skovgaard and Rudolph. Each cell division requires the complete and accurate duplication of the entire genome. In bacteria, the duplication process of the often-circular chromosomes is initiated at a single origin per chromosome, resulting in two replication forks that traverse the chromosome in opposite directions. DNA synthesis is completed once the two forks fuse in a region diametrically opposite the origin. In some bacteria, such as Escherichia coli, the region where forks fuse forms a specialized termination area. Polar replication fork pause sites flanking this area can pause the progression of replication forks, thereby allowing forks to enter but not to leave. Transcription of all required genes has to take place simultaneously with genome duplication. As both of these genome trafficking processes share the same template, conflicts are unavoidable. In this review, we focus on recent attempts to add additional origins into various ectopic chromosomal locations of the E. coli chromosome. As ectopic origins disturb the native replichore arrangements, the problems resulting from such perturbations can give important insights into how genome trafficking processes are coordinated and the problems that arise if this coordination is disturbed. The data from these studies highlight that head-on replication–transcription conflicts are indeed highly problematic and multiple repair pathways are required to restart replication forks arrested at obstacles. In addition, the existing data also demonstrate that the replication fork trap in E. coli imposes significant constraints to genome duplication if ectopic origins are active. We describe the current models of how replication fork fusion events can cause serious problems for genome duplication, as well as models of how such problems might be alleviated both by a number of repair pathways as well as the replication fork trap system. Considering the problems associated both with head-on replication- transcription conflicts as well as head-on replication fork fusion events might provide clues of how these genome trafficking issues have contributed to shape the distinct architecture of bacterial chromosomes.Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council, research grants BB/K015729/1 and BB/N014995/1

Origins left, right and centre: increasing the number of initiation sites in the Escherichia coli chromosome

© 2018 by the authors. The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication–transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY, integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replication–transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture

Cas1–Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation

Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chro-mosomal self-targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replica-tion, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA

Quinone transfer radical polymerization (QTRP) of styrene: Catalysis by different metal complexes

peer reviewedaudience: researcherStyrene has been polymerized by a Quinone Transfer Radical Polymerization (QTRP) based on the redox reaction of an ortho-quinone and a metal catalyst. Several metal acetylacetonates have been tested in this work. The radical polymerization of styrene is largely controlled when phenanthrenequinone (PhQ) is used with catalytic amounts of Co(acac)(2), Ni(acac)(2), Mn(acac)2 or 3, and Al(acac)(3). As a rule, in the presence of all these metallic complexes, the polystyrene molar mass increases with the monomer conversion, and polydispersity (M-w/M-n) is in the 1.3-1.6 range (at least until 40% monomer conversion). Styrene polymerization has also been resumed by polystyrene chains prepared by QTRP. In the specific case of manganese acetylacetonates, an amine or phosphine ligand has to be added for the control to be effective. Finally, two mechanistic hypotheses have been proposed, depending on whether the oxidation state of the metal can be easily changed or not

25 years on and no end in sight: a perspective on the role of RecG protein.

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced 'adaptive' mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.CJR is supported by a grant from the Biotechnology and Biological Sciences Research Council [BB/K015729/1]