DNA BENDING AND CONFORMATIONAL SWITCHING

University Of Washington, Seattle WA

Investigators

Linked publications & trials

Abstract

The long-term objectives are (1) to characterize the mechanisms of spontaneous and protein-induced flexure of DNA, (2) to elucidate insofar as possible the contribution of conformational switching to the bending process, and (3) to assess long-range effects of protein-induced bends on the secondary structure of DNA. Previous experiments indicate that induced bending strain alters the average secondary conformation and properties that reflect that. Recent measurements reveal that the bending rigidity relaxes from a rather high dynamic value, characterized by a dynamic persistence length (Pd), at short times (t less than or equal to 10-5 s) to an equilibrium value that is 40 percent or less of the dynamic value at long times. Both effects are attributed to a shift in a prevailing equilibrium between differently curved secondary conformations. Pd governs the flexural dynamics at short times, whereas the total persistence length (Ptot) reflects DNA curvature due to all kinds of bends. A principal theme of the proposed research is to assess Pd, Ptot, and the torsion elastic constant (alpha) by measuring the optical anisotropies of different DNAs and DNA-protein complexes from 0 to 120 ns by time-resolved fluorescence polarization anisotropy (FPA) and from 30 ns to 10 mus or more by a recently developed transient polarization grating (TPG) method. The relative ethidium binding constant will be obtained via the fluorescence decay kinetics and the circular dichroism (CD) spectrum will also be measured to provide additional probes of changes in secondary conformation. The plan is to perform FPA and TPG measurements on 181, 225, and 250 by DNAs, and to combine those with existing data on 200 by DNAs to test the length dependence of the model and to more robustly estimate Pd and alpha. Similarly, Pd, Ptot, and alpha and the CD spectra of 200 bp DNAs will be measured under various conditions to investigate the effects of (1) temperature over the premelting range 5-60 OC, (2) partial dehydration by neutral osmolytes, (3) overall percent GC composition, and (4) substitution of a 16 bp (CG)8 sequence near the middle. In addition, the effects of binding IHF to the ends of a 277 bp DNA will be studied to determine any effect of the large induced bends on the intervening DNA.