The Science Seminar Series: January 31, 2008 4pm

Dynamics of Loading the Escherichia coli DNA Polymerase Processivity Clamp onto DNA

Dr. Linda Bloom

Department of Biochemistry & Molecular Biology

University of Florida

Powell Hall

Time: 4:00 - 5:00pm

Abstract

Clamp loaders are molecular machines that catalyze the assembly of ring-shaped sliding clamps onto DNA to facilitate DNA replication. By encircling DNA and binding directly to DNA polymerases, sliding clamps effectively tether polymerases to the templates being copied. This increases the processivity of DNA synthesis, or number of nucleotides incorporated in a single DNA binding event, from tens of nucleotides to thousands of nucleotides. Using proteins from Escherichia coli as a model system, our laboratory is investigating the mechanism by which clamp loaders assemble clamps on DNA. This reaction is dynamic in that it requires protein•protein and protein•DNA interactions to change rapidly during the course of the reaction. Minimally, the clamp loader must have a high affinity for the clamp and DNA to bring these macromolecules together, but then the affinity must be reduced for the clamp loader to release the clamp on DNA for use by a DNA polymerase. ATP binding and hydrolysis contribute to this affinity modulation by promoting binding and release, respectively, of the clamp and DNA. However, ATP binding and hydrolysis alone are not sufficient to establish a temporal order of events that leads to the most efficient clamp loading reaction. Our hypothesis is that each interaction that the clamp loader makes with its binding partners including ATP, ADP + Pi, the clamp, and DNA promote conformational changes in the clamp loader that facilitate the next step in the loading reaction. Our laboratory has developed a number of real time fluorescence-based techniques to investigate the clamp loading reaction directly in solution and in real time to uncover the temporal order of events in the clamp loading reaction and define the mechanism of clamp loading. A combination of DNA binding, clamp binding, and ATP hydrolysis assays were used to define the order of events that occur when a clamp loader clamp complex binds DNA. These results will be discussed in terms of our model for clamp loading.