Dynamics of Elementary Processes in Solutions of Biopolymers

University Of Washington, Seattle WA

Investigators

Abstract

MCB 9982735 Schurr, J. M. The molecular mechanisms of DNA bending, the effects of decreasing salt concentration on the structures and thermodynamics of supercoiled plasmids, and the effects of the osmotic exclusion agent polyethylene glycol (PEG) on supercoiling free energies will be investigated by a variety of methods. Analytical theory, as well as Monte Carlo and Brownian Dynamics simulations, will be employed to extract the relevant parameters and assist the interpretation of results. A transition in secondary structure caused by the bending strain in 250-350 bp DNA circles will be investigated by measuring both the torsion elastic constants and ethidium binding constants (via fluorescence methods) and also the circular dichroism (CD) spectra of ~280 and ~320 bp circles. A newly developed transient polarization grating (TPG) instrument will be further refined and applied to assess the dynamic bending rigidity and equilibrium persistence length of ~175-250 bp linear DNAs. The contribution of slowly relaxing fluctuations between differently curved secondary structures to the equilibrium flexibility of DNA will be estimated from such measurements. Finally, the translational diffusion coefficients and static structure factors of supercoiled plasmids will be measured via dynamic and static light scattering (DLS and SLS), and compared with the results of Monte Carlo simulations to assess the structures and persistence lengths under low salt conditions. The effects of PEG on the supercoiling free energy will be assessed by standard topoisomer analysis methods. Possible effects of PEG on secondary structure will also be studied by fluorescence and CD methods. DNA is bent and also twisted in most or all of its biologically important states, including its supercoiled states and its complexes with transcriptional activators. Detailed knowledge of the nature and energetics of bending is essential to understand the function and stability of such states. In this work, structural transitions induced by the bending of short linear DNAs into small circles, and also by the supercoiling of long DNAs at low salt concentration, will be studied to illuminate the process wherein bending alters the secondary structure of the DNA.