Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131-151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms

Single-Fluorophore Diffusion in a Lipid Membrane over a Subwavelength Aperture

We use submicrometer apertures milled in an aluminium film to study the diffusion dynamics of β-Bodipy-FL-C5-HPC (Bodipy-PC) fluorophores in a lipid dioleoylphosphatidylcholine (DOPC) multilayer. The observation volume is limited by the aperture diameter, well below the optical wavelength. This spatial resolution improvement comes together with an enhancement of the detected fluorescence per molecule as compared to an open sample, with a significant increase up to 3.5 times

Regulatory Requirements Traceability and Analysis Using Semi-Formal Specifications

Abstract: Information systems are increasingly distributed and pervasive, enabling organizations to deliver remote services and share personal information, worldwide. However, developers face significant challenges in managing the many laws that govern their systems in this multi-jurisdictional environment. In this paper, we report on a computational requirements document expressible using a legal requirements specification language (LRSL). The purpose is to make legal requirements open and available to policy makers, business analysts and software developers, alike. We show how requirements engineers can codify policy and law using the LRSL and design, debug, analyze, trace, and visualize relationships among regulatory requirements. The LRSL provides new constructs for expressing distributed constraints, making regulatory specification patterns visually salient, and enabling metrics to quantitatively measure different styles for writing legal and policy documents. We discovered and validated the LRSL using thirteen U.S. state data breach notification laws