The long-term goal of Dr. Fischer's research is to obtain a quantitative molecular understanding of the mechanism(s) of ATP-dependent chromatin regulation in Eukaryotes. The specific objective of this proposed project is the quantitative characterization of the kinetics of ATP-dependent DNA translocation by the RSC chromatin remodeling enzyme from S. cerevisiae. Even though several proposed models of ATPdependent chromatin remodeling require that the remodeling enzyme translocate along DNA, there has not
yet been a complete and quantitative kinetic study of the DNA translocation mechanism of any remodeling enzyme.

Fischer's proposed studies are therefore novel and will provide considerable insight into the behavior of RSC other chromatin remodeling complexes. The DNA translocation activity of RSC will be primarily monitored through measurements of the DNA-stimulated ATPase activity of the enzyme using standard radioactivity-based assays. As demonstrated in his group's preliminary results, they can analyze the time course of this ATPase activity to determine potential kinetic models of DNA translocation and estimates of the associated processivities. Further refinement of these models including estimates of additional kinetic parameters will be accomplished through analysis of DNA translocation time courses obtained using well-established stopped-flow fluorescence-based assays. In these experiments the position of the translocating protein on the DNA is inferred from changes in the fluorescence of fluorophores attached to the DNA resulting from interaction with the translocating protein. Taken together, these data will also allow for the calculation of the
thermodynamic efficiency (ATP coupling stoichiometry) of the molecular motor driving RSC translocation.