Predoctoral Fellowship for Sarah Katharine Ponticelli-Mechanism for Loading the E.Coli Beta Clamp on DNA

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how organisms coordinate the replication and repair of their genomes. The use of processivity clamps that must be loaded onto DNA during replication of chromosomes by an ATP dependent clamp loader complex is highly conserved by all forms of life. Understanding how organisms coordinate DNA replication and repair is the fundamental basis for understanding human diseases such as cancer. The goal of the work proposed here is to better define the mechanism of clamp loading in the E. coli model system. The E. coli clamp loader (gamma complex) is a multisubunit complex that undergoes a conformational change upon binding ATP which facilitates opening of the beta clamp and its subsequent loading onto DNA. The contacts of the individual clamp loader subunits with beta differ between the ATP bound and free states of gamma complex. The interactions of the gamma complex subunits with beta in both the presence and absence of ATP will be determined. Biochemical assays using various clamp and clamp loader mutants will be utilized to accomplish this. The insight gained from E. coli can provide a framework for elucidating the mechanisms of eukaryotes, including humans.