Geometry and mechanics of DNA superhelicity

This paper analyzes the elastic equilibrium conformations of duplex DNA constrained by the constancy of its molecular linking number, Lk. The DNA is regarded as having the mechanical properties of a homogeneous, linearly elastic substance with symmetric cross section. Integral representations of the writhing number Wr and of Lk are developed, in terms of which the equilibria are given as solutions to an isoperimetric problem. It is shown that the Euler angles defining equilibrium conformations must obey equations identical to those governing unconstrained equilibria. A scaling law is developed stating that molecules supercoiled the same amount ΔLk will have geometrically similar elastic equilibria regardless of their length. Thus, comparisons among molecules of properties related to their large-scale tertiary structure should be referred to differences in ΔLk rather than to their superhelix densities. Specific conditions on the elastic equilibrium conformations are developed that are necessary for ring closure. The equilibrium superhelical conformations accessible to closed-ring molecules are shown to approximate toroidal helices. Questions relating to the stability and nonuniqueness of equilibria are treated briefly. A comparison is made between these toroidal conformations and interwound configurations, which are shown to be stable, although they are not equilibria in the present sense. It is suggested that entropic factors are responsible for favouring the toroidal conformation in solution.     

Effect of DNA superhelicity and bound proteins on mechanistic aspects of the Hin-mediated and Fis-enhanced inversion

Using a recently developed inhomogeneous, macroscopic model for long DNA bound to proteins, we examine topological and geometric aspects of DNA/protein structures and dynamics on various stages of the Hin inversion pathway. This biological reaction involves exchange of DNA in a synaptic complex that brings together several DNA sites bound to Hin dimers as well as Fis enhancers. Brownian dynamics simulations in the millisecond timescale allow us to follow and analyze the DNA/protein dynamics trajectories and to examine the effects of DNA superhelicity and protein binding on various reaction steps. Analysis of the generated kinetic pathways helps explain mechanistic aspects regarding the process by which two or three protein-bound DNA sites come to close spatial proximity and show that how topological selectivity (two trapped supercoils), enhancer binding, and properties of supercoiled DNA play critical roles in regulating the inversion reaction. Specifically, a critical amount of DNA superhelicity (e.g., |sigma| > 0.02) leads to an optimal interplay for the first reaction step-two-site juxtaposition-between large-scale random rearrangements of Hin-bound DNA and local slithering within branches of plectonemes. The three-site juxtaposition, the second step, is significantly accelerated by the presence of an enhancer protein that, due to severe local bending, also alters juxtaposition mechanisms, especially for superhelical density magnitude greater than around 0.04.     

Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus

Summary The thermostability of the staphylococcal plasmids pC194 and pUB110 and their antibiotic-resistance determinants was examined upon transfer to Bacillus stearothermophilus CU21. Plasmid pGS13, a pUB110 derivative carrying the chloramphenicol acetyltransferase (CAT) gene of pC194, could be maintained up to the maximum growth temperature (68° C) by selection for chloramphenicol resistance. In the absence of selective pressure, pGS13 was lost at temperatures above 60° C. Segregational instability of pGS13 was accompanied by a progressive loss of negative superhelicity at elevated temperatures. Thermostable mutants of pGS13 were isolated by screening for expression of the antibiotic-resistance determinants after growth under non-selective conditions. These mutants were found to contain an insertion of a 1.7 kb DNA sequence derived from the cryptic B. stearothermophilus plasmid pBS02. Increased thermostability correlated with preservation of plasmid superhelicity at elevated temperatures.