SSB Protein/DNA Interactions

Washington University, Saint Louis MO

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Abstract

DESCRIPTION (provided by applicant): Single Stranded Binding (SSB) Proteins are central players in DNA replication, recombination and repair due to their interactions with DNA and myriad accessory proteins. We seek a molecular understanding of the interactions of the E. coli EcoSSB, and the human RPA proteins, with single stranded (ss) DNA and accessory proteins. Ensemble thermodynamic and kinetic approaches, as well as novel single molecule approaches will be used to investigate the energetics and dynamics of these interactions. EcoSSB serves as a paradigm for a growing number of similar proteins possessing multiple DNA binding domains, displaying 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 binding modes differ in the extent to which ssDNA is wrapped around the tetramer and we hypothesize that they are used selectively in different processes in vivo. Our extensive knowledge of the EcoSSB protein makes it one of the best systems for continued studies of the thermodynamic profile of a protein-ssDNA binding system. The thermodynamics of ssDNA binding will be examined by isothermal titration calorimetry (ITC) and Differential Scanning Calorimetry (DSC), focusing on the contributions of protein conformational changes to the extremely large ?Hobs and the dramatic changes in ?CP,obs on salt concentration and temperature. A new major focus is to understand the dynamics of spontaneous wrapping/unwrapping of ssDNA around the SSB tetramer and to test for possible protein movement along ssDNA by a "rolling" mechanism using single molecule fluorescence techniques, Fluorescence Correlation Spectroscopy (PCS) and stopped-flow. Optical tweezer approaches will be used to examine the effects of the different SSB binding modes on RecA binding to SSB-coated ssDNA. Our studies of the RPA protein have shown that it also binds ssDNA in multiple binding modes and we will continue these studies. The effects of the different SSB-ssDNA modes on the specificity of EcoSSB binding to a variety of replication proteins will also be studied, as well as the kinetic mechanism of duplex DNA melting by these SSB proteins.