SSB PROTEIN/DNA INTERACTIONS

Washington University, Saint Louis MO

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Abstract

DESCRIPTION (provided by applicant): A molecular understanding is sought of the interactions of the Single Stranded DNA Binding (SSB) proteins from E. coli the homo-tetrameric EcoSSB, and yeast, the hetero-trimeric RPA; with single stranded (ss) DNA using thermodynamic, kinetic, single molecule and structural approaches. Both of these proteins bind selectively to ssDNA and are essential for DNA replication and repair and facilitate recombination. EcoSSB serves as a paradigm for a growing number of similar proteins from other organisms. EcoSSB displays a complex array of multiple ssDNA binding modes, multiple inter-tetramer positive cooperativities and a negative cooperativity for ssDNA binding within an individual tetramer that regulates the binding modes. These different binding modes, which differ in the extent to which DNA is wrapped around the tetramer, are likely used selectively in different processes in vivo. The EcoSSB protein also serves as a model to understand the fundamental thermodynamic profile of a proteinssDNA binding system. A major goal is to understand the effects of salt concentration on the thermodynamics and kinetics, since electrostatic effects are a prominent component of protein-nucleic acid systems. The thermodynamics of oligodeoxynucleotide binding will be examined by isothermal titration calorimetry (ITC), to obtain deltaGxobs, deltaHobs, deltaSxobs and deltaCP, obs as a function of solution conditions, focussing on the extremely large and favorable deltaHobs and the dramatic dependence of deltaHobs and deltaCP, obs on salt concentration. The thermodynamics of cooperative ssDNA binding in both the (SSB)35 and (SSB)65 binding modes will be examined and single molecule fluorescence techniques will be used to examine ssDNA wrapping. Kinetic mechanisms of ssDNA binding, ssDNA wrapping and "direct transfer" of SSB between ssDNA molecules will be examined using fluorescence stopped-flow and temperature-jump techniques. The energetics of EcoSSB binding to replication proteins such as the chi subunit of the gamma complex of DNA pol III will be pursued. High resolution structural information will also be sought on complexes of EcoSSB with ssDNA and the X subunit through x-ray crystallographic analysis in order to place the thermodynamic studies in a structural context. Finally, thermodynamic studies of ssDNA binding to the hetero-trimeric RPA protein, the eukaryotic analog of EcoSSB, will be pursued to examine its ability to bind in multiple binding modes, and compare its properties to those of EcoSSB.