DNA translocation by type III restriction enzymes: a comparison of current models of their operation derived from ensemble and single-molecule measurements

Much insight into the interactions of DNA and enzymes has been obtained using a number of single-molecule techniques. However, recent results generated using two of these techniques—atomic force microscopy (AFM) and magnetic tweezers (MT)—have produced apparently contradictory results when applied to the action of the ATP-dependent type III restriction endonucleases on DNA. The AFM images show extensive looping of the DNA brought about by the existence of multiple DNA binding sites on each enzyme and enzyme dimerisation. The MT experiments show no evidence for looping being a requirement for DNA cleavage, but instead support a diffusive sliding of the enzyme on the DNA until an enzyme–enzyme collision occurs, leading to cleavage. Not only do these two methods appear to disagree, but also the models derived from them have difficulty explaining some ensemble biochemical results on DNA cleavage. In this ‘Survey and Summary’, we describe several different models put forward for the action of type III restriction enzymes and their inadequacies. We also attempt to reconcile the different models and indicate areas for further experimentation to elucidate the mechanism of these enzymes

Proposing a rational resilience credo for use with athletes

© 2016 Association for Applied Sport Psychology. While the reported use of Rational Emotive Behavior Therapy (REBT) is growing in sport, little is written about specific tools used by practitioners when applying REBT with athletes. The Athlete Rational Resilience Credo (ARRC) adapts Windy Dryden's (2007) original Rational Resilience Credo for application with athletes. The ARRC promotes rational beliefs in athletes, which are important for resilient responding to adverse events. The ARRC is presented in full, followed by some explanation as to its purposes, critical practitioner reflections, and guidance for its use in sport

The Bayesian Spatial Bradley--Terry Model: Urban Deprivation Modeling in Tanzania

Identifying the most deprived regions of any country or city is key if policy makers are to design successful interventions. However, locating areas with the greatest need is often surprisingly challenging in developing countries. Due to the logistical challenges of traditional household surveying, official statistics can be slow to be updated; estimates that exist can be coarse, a consequence of prohibitive costs and poor infrastructures; and mass urbanisation can render manually surveyed figures rapidly out-of-date. Comparative judgement models, such as the Bradley--Terry model, offer a promising solution. Leveraging local knowledge, elicited via comparisons of different areas' affluence, such models can both simplify logistics and circumvent biases inherent to house-hold surveys. Yet widespread adoption remains limited, due to the large amount of data existing approaches still require. We address this via development of a novel Bayesian Spatial Bradley--Terry model, which substantially decreases the amount of data comparisons required for effective inference. This model integrates a network representation of the city or country, along with assumptions of spatial smoothness that allow deprivation in one area to be informed by neighbouring areas. We demonstrate the practical effectiveness of this method, through a novel comparative judgement data set collected in Dar es Salaam, Tanzania.Comment: 23 pages, 7 figures, to be published in the journal of the Royal Statistical Society: Series

Crater lake cichlids individually specialize along the benthic-limnetic axis

A common pattern of adaptive diversification in freshwater fishes is the repeated evolution of elongated open water (limnetic) species and high-bodied shore (benthic) species from generalist ancestors. Studies on phenotype-diet correlations have suggested that population-wide individual specialization occurs at an early evolutionary and ecological stage of divergence and niche partitioning. This variable restricted niche use across individuals can provide the raw material for earliest stages of sympatric divergence. We investigated variation in morphology and diet as well as their correlations along the benthic-limnetic axis in an extremely young Midas cichlid species, Amphilophus tolteca, endemic to the Nicaraguan crater lake Asososca Managua. We found that A. tolteca varied continuously in ecologically relevant traits such as body shape and lower pharyngeal jaw morphology. The correlation of these phenotypes with niche suggested that individuals are specialized along the benthic-limnetic axis. No genetic differentiation within the crater lake was detected based on genotypes from 13 microsatellite loci. Overall, we found that individual specialization in this young crater lake species encompasses the limnetic- as well as the benthic macro-habitat. Yet there is no evidence for any diversification within the species, making this a candidate system for studying what might be the early stages preceding sympatric divergence

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPγS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping

Transition in incompressible boundary layers with two-dimensional excrescences

An experimental investigation of the transition process in boundary layers subjected to forward- or aft-facing two-dimensional step excrescences is described. The objective of the work was to characterize the variation of transition Reynolds numbers with measurable roughness and boundary layer parameters, with the specific goal of specifying new tolerance criteria for laminar flow airfoils, alongside a fundamental investigation of linear boundary layer stability mechanisms. Results from an ongoing program of increasing complexity on effects of pressure gradient on excrescence-induced transition are presented. Preliminary N-factor calculations are used to determine the effects of boundary layer stability and attempt to isolate the effect of the disturbance due to the excrescence

Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy

AbstractBacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA

Classical and quantum ergodicity on orbifolds

We extend to orbifolds classical results on quantum ergodicity due to Shnirelman, Colin de Verdi\`ere and Zelditch, proving that, for any positive, first-order self-adjoint elliptic pseudodifferential operator P on a compact orbifold X with positive principal symbol p, ergodicity of the Hamiltonian flow of p implies quantum ergodicity for the operator P. We also prove ergodicity of the geodesic flow on a compact Riemannian orbifold of negative sectional curvature.Comment: 14 page

On the stationarity of linearly forced turbulence in finite domains

A simple scheme of forcing turbulence away from decay was introduced by Lundgren some time ago, the `linear forcing', which amounts to a force term linear in the velocity field with a constant coefficient. The evolution of linearly forced turbulence towards a stationary final state, as indicated by direct numerical simulations (DNS), is examined from a theoretical point of view based on symmetry arguments. In order to follow closely the DNS the flow is assumed to live in a cubic domain with periodic boundary conditions. The simplicity of the linear forcing scheme allows one to re-write the problem as one of decaying turbulence with a decreasing viscosity. Scaling symmetry considerations suggest that the system evolves to a stationary state, evolution that may be understood as the gradual breaking of a larger approximate symmetry to a smaller exact symmetry. The same arguments show that the finiteness of the domain is intimately related to the evolution of the system to a stationary state at late times, as well as the consistency of this state with a high degree of isotropy imposed by the symmetries of the domain itself. The fluctuations observed in the DNS for all quantities in the stationary state can be associated with deviations from isotropy. Indeed, self-preserving isotropic turbulence models are used to study evolution from a direct dynamical point of view, emphasizing the naturalness of the Taylor microscale as a self-similarity scale in this system. In this context the stationary state emerges as a stable fixed point. Self-preservation seems to be the reason behind a noted similarity of the third order structure function between the linearly forced and freely decaying turbulence, where again the finiteness of the domain plays an significant role.Comment: 15 pages, 7 figures, changes in the discussion at the end of section VI, formula (60) correcte