Dynamics of Protein-DNA Interactions in DNA Replication

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DESCRIPTION (provided by applicant) Functional themes in replicases have been conserved from prokaryotes to eukaryotes. DNA polymerases use ring-shaped sliding clamps to achieve the processivity required to replicate a genome. There are remarkable structural similarities in sliding clamps from bacteria, yeast, and man. These clamps are assembled onto DNA by the activity of multisubunit clamp loaders that couple hydrolysis of ATP to the mechanical task of clamp loading. This process requires that protein-protein and protein-DNA interactions change during the clamp loading reaction. Binding and hydrolysis of ATP promote conformational changes in the clamp loader that modulate these interactions. We propose to define the dynamic protein-protein and protein-DNA interactions that are required for the gamma complex clamp loader to assemble the Escherichia coil DNA polymerase III beta clamp onto DNA. Real time fluorescence-based assays will be used to monitor protein-protein and protein-DNA interactions directly in solution and correlate these with ATP binding and hydrolysis. This approach gives our laboratory the unique ability to define changes in the protein-protein and protein-DNA interactions that occur on a millisecond timescale during the clamp loading reaction. We propose to use these experiments to test a kinetic model for clamp loading by defining interactions between the clamp loader and clamp, by defining nucleotide-dependent steps, and by using FRET to measure clamp opening and closing. Much of what has been learned about these replisomes has come from studies of the E. coli system and many analogies are seen in the human system. An E. coil model system offers the advantage that stable proteins can be obtained in high yields for in vitro analysis.