Abstract:

Replication protein A (RPA) coordinates a plethora of DNA metabolic events. In the cell, it binds to virtually all exposed single-strand DNA, melts secondary DNA structures, recruits over three dozen proteins onto ssDNA,  activates the DNA damage response, and hands off ssDNA to appropriate downstream players. All these activities depend on the dynamic binding and dissociation of the four individual DNA binding domains (DBDs). To visualize and quantify the DBDs dynamics, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM), biophysical and biochemical analyses. In this talk, I will describe how microscopic dynamics of DBDs in the context of the macroscopically bound RPA promotes RPA replacement with lower affinity DNA binding proteins, and how this dynamics is regulated in homologous recombination and during maintenance of human telomeres.