This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this work is to determine the molecular details of DNA structure regulation and as a result to understand why mutations in human proteins responsible for this regulation result in the development of cancer. The information obtained from this project will be useful in the development of drugs which will specifically prevent these proteins from malfunctioning and thereby causing disease. The long-term goal of this 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 ATP-dependent 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. My proposed novel studies 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 our preliminary results, we 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.
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