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

Observation of one-way Einstein-Podolsky-Rosen steering

The distinctive non-classical features of quantum physics were first discussed in the seminal paper by A. Einstein, B. Podolsky and N. Rosen (EPR) in 1935. In his immediate response E. Schr\"odinger introduced the notion of entanglement, now seen as the essential resource in quantum information as well as in quantum metrology. Furthermore he showed that at the core of the EPR argument is a phenomenon which he called steering. In contrast to entanglement and violations of Bell's inequalities, steering implies a direction between the parties involved. Recent theoretical works have precisely defined this property. Here we present an experimental realization of two entangled Gaussian modes of light by which in fact one party can steer the other but not conversely. The generated one-way steering gives a new insight into quantum physics and may open a new field of applications in quantum information.Comment: 4 pages, 4 figure

Creationism and Intelligent Design

Until recently, little attention has been paid in the school classroom to creationism and almost none to intelligent design. However, creationism and intelligent design appear to be on the increase and there are indications that there are more countries in which schools are becoming battlegrounds over them. I begin by examining whether creationism and intelligent design are controversial issues, drawing on Robert Dearden’s epistemic criterion of the controversial and more recent responses to and defences of this. I then examine whether the notion of ‘worldviews’ in the context of creationism is a useful one by considering the film March of the Penguins. I conclude that the ‘worldviews’ perspective on creationism is useful for two reasons: first, it indicates the difficulty of using the criterion of reason to decide whether an issue is controversial or not; secondly, it suggests that standard ways of addressing the diversity of student views in a science classroom may be inadequate. I close by examining the implications of this view for teaching in science lessons and elsewhere, for example in religious education lessons and at primary level where subject divisions cannot be made in so clear-cut a manner