E COLI SSB PROTEIN/DNA INTERACTIONS

Washington University, Saint Louis MO

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Abstract

The proposed research is to obtain a molecular understanding of the interactions of the E. coli Single Stranded DNA Binding (SSB) protein with single stranded (ss) DNA using thermodynamic, kinetic and structural approaches. The SSB protein is a helix destabilizing protein that binds selectively and cooperatively to ss-polynucleotides and is essential DNA replication and repair and facilitates homologous recombination in E. coli. It is a homo-tetramer and serves as a paradigm for a growing number of similar tetrameric SSB proteins from other organisms, including human mitochondria. The protein displays a complex array of ss-DNA binding features, including multiple DNA binding modes, multiple inter-tetramer positive cooperativities, and dramatic negative cooperativity for ss-DNA binding within an individual tetramer. There is evidence suggesting that the different SSB-ss-polynucleotide binding modes and cooperativities may be used selectively in different functions in vivo and that the transition between these modes is regulated by the negative cooperativity. We are interested in the SSB protein since it is an essential DNA replication protein and a paradigm for the class of SSB proteins. However, we also study it to probe the fundamental thermodynamic linkages that occur in protein-DNA interactions and as a model for studies of negative cooperativity in protein-ligand systems. Detailed thermodynamic studies over a range of solution conditions are essential to obtain an understanding of the functional energetics of protein-DNA interactions. A particular emphasis of our studies is to probe the thermodynamic effects of salt concentration and its linkage to other variable, since electrostatic effects are dominant in protein-nucleic acid systems. The proposed studies focus on SSB-oligodeoxynucleotide interactions since these allow more precise determinations of the thermodynamics of binding and negative cooperativity. The thermodynamics of oligodeoxynucleotide binding will be examined by two approaches: (1) equilibrium binding parameters (binding and negative cooperativity constants) will be obtained as a function of solution conditions (pH, temperature, salt concentration and type) from analyses of equilibrium isotherms obtained by fluorescence techniques and (2) isothermal titration calorimetry. Kinetic and mechanistic studies will be performed using fluorescence stopped-flow techniques. We will also initiate a new project to examine the ability of the SSB protein to destabilize intramolecular hairpins that can form within ss-DNA during replication and in transiently melted regions of duplex DNA. We are also collaborating wit Dr. Gabriel Waksman to obtain atomic level structural information through X-ray crystallographic analysis of co-crystals of SSB-ss-oligodeoxynucleotide complexes that we have recently obtained.