This project transferred from LCB. The former project number was Z01 DK 2505003 LCB. Major emphasis has been placed on identifying and overcoming the problems associated with photochemical electric dichroism. This technique is a hybrid of classical electric dichroism and the methodologies developed by molecular biologists for examining DNA- protein complexes. The end result will be a technique that can sensitively and selectively map the topology of DNA folding or wrapping in specific DNA-protein complexes of biological importance. The problems we have encountered primarily result from the very large electric fields applied across the sample and the very intense UV laser light that must be employed. Electrode poisoning is the primary effect. A greater emphasis on electrode cleaning and preparation appear essential for optimal results. Additionally, buffer conditions for the dichroism experiment are far more critical for this type of experiment than classical dichroism because of non specific background nicking of DNA. improvements in signal quality and differences observed in photodamage probability between oriented and unoriented reconstituted nucleosomes indicate that the technique with work and become a dominant structural technique for protein-DNA complexes with sensitivities and specificities comparable to footprinting. We have installed a new computer-linked data collection system to our electric birefringence and dichroism apparatuses. The LeCroy system gives a greater time resolution and makes data analysis faster and more convenient. Work or other projects can now proceed at a faster rate. We have expanded our work on bent DNA fragments to include oligo A tract sequences that show interesting flanking sequence properties. Initial results or bipolar myosis filaments indicate a great deal of internal motion and that this motion is different for phosphorylated and unphosphorylated myosin. Building on our previous results we are about ready to construct dynamical models for the bipolar filament.
