The consequences of replicating in the wrong orientation: Bacterial chromosome duplication without an active replication origin

Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid meta¬bolism. In cells lacking RNase HI replication initiates indepen¬dently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly-transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics and, ultimately, cell viability.This work was supported by the Royal Society (RG110414 to C.J.R.) and The Biotechnology and Biological Sciences Research Council (BB/K015729/1 to C.J.R.)

Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD

and proceed in opposite directions with high speed and processivity until they fuse and terminate 19 in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound 20 protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the 21 RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. 22 It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of 23 replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA 24 degradation in the termination area. In this study we show that head-on encounters of replication 25 and transcription at a highly-transcribed rrn operon expose fork structures to degradation by 26 nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination 27 area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this 28 location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two 29 different locations highlight that the location of replication termination can have some impact on 30 the degradation observed. Our data improve our understanding of the role of RecBCD at sites of 31 replication-transcription conflicts as well as the final stages of chromosome duplication. 32 However, they also highlight that current models are insufficient and cannot explain all the 33 molecular details in cells lacking RecBCD

Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed

Chromosome duplication initiates via the assembly of replication forks at defined origins. Forks proceed in opposite directions until they fuse with a converging fork. Recent work highlights that fork fusions are highly choreographed both in pro- and eukaryotic cells. The circular Escherichia coli chromosome is replicated from a single origin (oriC), and a single fork fusion takes place in a specialised termination area opposite oriC that establishes a fork trap mediated by Tus protein bound at ter sequences that allows forks to enter but not leave. Here we further define the molecular details of fork fusions and the role of RecG helicase in replication termination. Our data support the idea that fork fusions have the potential to trigger local re-replication of the already replicated DNA. In ΔrecG cells this potential is realised in a substantial fraction of cells and is dramatically elevated when one fork is trapped for some time before the converging fork arrives. They also support the idea that the termination area evolved to contain such over-replication and we propose that the stable arrest of replication forks at ter/Tus complexes is an important feature that limits the likelihood of problems arising as replication terminates

Clearing transcription barriers to replication

Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote forkmovement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of fulllength replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.UK Biotechnology and Biological Sciences Research Council (BBSRC) [BB/I001859/2, BB/N014863/1 to P.M., BB/K015729/1, BB/N014995/1 to C.J.R. and BB/I003142/1 to N.J.S. and M.S.D.]. Funding for open access charge: York Open Access Fund

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

Data Availability: The data underlying this article are available in the article and in its online supplementary material....BBSRC grant number BB/T006625-1 (ELB) and BB/T007168/1 (CJR); Croatian Science Foundation grant IP-2016-06-8861 (IIB)

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]