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.)

Mathematical modeling of genome replication

Peer reviewedPublisher PD

The dynamics of genome replication using deep sequencing

Peer reviewedPublisher PD

Mathematical modelling of whole chromosome replication

All chromosomes must be completely replicated prior to cell division, a requirement that demands the activation of a sufficient number of appropriately distributed DNA replication origins. Here we investigate how the activity of multiple origins on each chromosome is coordinated to ensure successful replication. We present a stochastic model for whole chromosome replication where the dynamics are based upon the parameters of individual origins. Using this model we demonstrate that mean replication time at any given chromosome position is determined collectively by the parameters of all origins. Combining parameter estimation with extensive simulations we show that there is a range of model parameters consistent with mean replication data, emphasising the need for caution in interpreting such data. In contrast, the replicated-fraction at time points through S phase contains more information than mean replication time data and allowed us to use our model to uniquely estimate many origin parameters. These estimated parameters enable us to make a number of predictions that showed agreement with independent experimental data, confirming that our model has predictive power. In summary, we demonstrate that a stochastic model can recapitulate experimental observations, including those that might be interpreted as deterministic such as ordered origin activation times

Image-based 3D canopy reconstruction to determine potential productivity in complex multi-species crop systems

Background and Aims: Intercropping systems contain two or more species simultaneously in close proximity. Due to contrasting features of the component crops, quantification of the light environment and photosynthetic productivity is extremely difficult. However it is an essential component of productivity. Here, a low-tech but high resolution method is presented that can be applied to single and multi-species cropping systems, to facilitate characterisation of the light environment. Different row layouts of an intercrop consisting of Bambara groundnut (Vigna subterranea (L.) Verdc.) and Proso millet (Panicum miliaceum) have been used as an example and the new opportunities presented by this approach have been analysed. Methods: Three-dimensional plant reconstruction, based on stereocameras, combined with ray-tracing was implemented to explore the light environment within the Bambara groundnut-Proso millet intercropping system and associated monocrops. Gas exchange data was used to predict the total carbon gain of each component crop. Key Results: The shading influence of the tall Proso millet on the shorter Bambara groundnut results in a reduction in total canopy light interception and carbon gain. However, the increased leaf area index (LAI) of Proso millet, higher photosynthetic potential due to the C4 pathway and sub-optimal photosynthetic acclimation of Bambara groundnut to shade means that increasing the number of rows of millet will lead to greater light interception and carbon gain per unit ground area, despite Bambara groundnut intercepting more light per unit leaf area. Conclusions: Three-dimensional reconstruction combined with ray tracing provides a novel, accurate method of exploring the light environment within an intercrop that does not require difficult measurements of light interception and data-intensive manual reconstruction, especially for such systems with inherently high spatial possibilities. It provides new opportunities for calculating potential productivity within multispecies cropping systems; enables the quantification of dynamic physiological differences between crops grown as monoculture and those within intercrops or; enables the prediction of new productive combinations of previously untested crops

Stochastic association of neighboring replicons creates replication factories in budding yeast

Peer reviewedPublisher PD

Three-dimensional plant architecture and sunlit-shaded patterns: a stochastic model of light dynamics in canopies

Background and Aims Diurnal changes in solar position and intensity combined with the structural complexity of plant architecture result in highly variable and dynamic light patterns within the plant canopy. This affects productivity through the complex ways that photosynthesis responds to changes in light intensity. Current methods to characterise light dynamics, such as ray-tracing, are able to produce data with excellent spatio-temporal resolution but are computationally intensive and the resultant data are complex and high dimensional. This necessitates development of more economical models for summarising the data and for simulating realistic light patterns over the course of a day. Methods High-resolution reconstructions of field-grown plants are assembled in various configurations to form canopies, and a forward ray-tracing algorithm is applied to the canopies to compute light dynamics at high (1 minute) temporal resolution. From the ray-tracer output, the sunlit or shaded state for each patch on the plants is determined, and these data are used to develop a novel stochastic model for the sunlit-shaded patterns. The model is designed to be straightforward to fit to data using maximum likelihood estimation, and fast to simulate from. Key Results For a wide range of contrasting 3D canopies, the stochastic model is able to summarise, and replicate in simulations, key features of the light dynamics. When light patterns simulated from the stochastic model are used as input to a model of photoinhibition, the predicted reduction in carbon gain is similar to that from calculations based on the (extremely costly) ray-tracer data. Conclusions The model provides a way to summarise highly complex data in a small number of parameters, and a cost-effective way to simulate realistic light patterns. Simulations from the model will be particularly useful for feeding into larger-scale photosynthesis models for calculating how light dynamics affects the photosynthetic productivity of canopies

High-resolution three-dimensional structural data quantify the impact of photoinhibition on long-term carbon gain in wheat canopies in the field

Photoinhibition reduces photosynthetic productivity; however, it is difficult to quantify accurately in complex canopies partly because of a lack of high-resolution structural data on plant canopy architecture, which determines complex fluctuations of light in space and time. Here, we evaluate the effects of photoinhibition on long-term carbon gain (over 1 d) in three different wheat (Triticum aestivum) lines, which are architecturally diverse. We use a unique method for accurate digital three-dimensional reconstruction of canopies growing in the field. The reconstruction method captures unique architectural differences between lines, such as leaf angle, curvature, and leaf density, thus providing a sensitive method of evaluating the productivity of actual canopy structures that previously were difficult or impossible to obtain. We show that complex data on light distribution can be automatically obtained without conventional manual measurements. We use a mathematical model of photosynthesis parameterized by field data consisting of chlorophyll fluorescence, light response curves of carbon dioxide assimilation, and manual confirmation of canopy architecture and light attenuation. Model simulations show that photoinhibition alone can result in substantial reduction in carbon gain, but this is highly dependent on exact canopy architecture and the diurnal dynamics of photoinhibition. The use of such highly realistic canopy reconstructions also allows us to conclude that even a moderate change in leaf angle in upper layers of the wheat canopy led to a large increase in the number of leaves in a severely light-limited state

The 4-dimensional plant: effects of wind-induced canopy movement on light fluctuations and photosynthesis

Physical perturbation of a plant canopy brought about by wind is a ubiquitous phenomenon and yet its biological importance has often been overlooked. This is partly due to the complexity of the issue at hand: wind-induced movement (or mechanical excitation) is a stochastic process which is difficult to measure and quantify; plant motion is dependent upon canopy architectural features which, until recently, were difficult to accurately represent and model in 3-dimensions; light patterning throughout a canopy is difficult to compute at high-resolutions, especially when confounded by other environmental variables. Recent studies have reinforced the expectation that canopy architecture is a strong determinant of productivity and yield; however, links between the architectural properties of the plant and its mechanical properties, particularly its response to wind, are relatively unknown. As a result, biologically relevant data relating canopy architecture, light- dynamics, and short-scale photosynthetic responses in the canopy setting are scarce. Here, we hypothesize that wind-induced movement will have large consequences for the photosynthetic productivity of our crops due to its influence on light patterning. To address this issue, in this study we combined high resolution 3D reconstructions of a plant canopy with a simple representation of canopy perturbation as a result of wind using solid body rotation in order to explore the potential effects on light patterning, interception, and photosynthetic productivity. We looked at two different scenarios: firstly a constant distortion where a rice canopy was subject to a permanent distortion throughout the whole day; and secondly, a dynamic distortion, where the canopy was distorted in incremental steps between two extremes at set time points in the day. We find that mechanical canopy excitation substantially alters light dynamics; light distribution and modeled canopy carbon gain. We then discuss methods required for accurate modeling of mechanical canopy excitation (here coined the 4-dimensional plant) and some associated biological and applied implications of such techniques. We hypothesize that biomechanical plant properties are a specific adaptation to achieve wind-induced photosynthetic enhancement and we outline how traits facilitating canopy excitation could be used as a route for improving crop yield