DNA Conformational Dynamics in Mismatch Recognition

Abstract

DNA mismatch recognition is done by the homodimer MutS in prokaryotes and by its homologues: heterodimers Msh2-Msh3 and Msh2-Msh6 in eukaryotes. Msh2-Msh6 binds preferentially to single insertion/deletions. Msh2-Msh3 has been shown to bind to DNA hairpins. It has been suggested that the conformational dynamics of the DNA substrate (bending and unbending) plays a fundamental role in the recognition process. Mismatch recognition allows identifying a single mismatched DNA pair among thousands of matched basepairs. The process is ATP dependent and different models for DNA discrimination have been proposed based in biochemical evidence as well as AFM studies. In this work we study the conformational dynamics of several DNA substrates and its complexes with the human MutS homologs. The DNA substrates were labeled with fluorescent dyes that constitute a fluorescence resonant energy transfer (FRET) pair. Experiments at the single molecule level allow us to follow the conformational dynamics of the substrates by determining the substrate's end to end distance. We were able to determine the binding and dissociation rates of the proteins from the substrates as well as the conformational state of the substrates under different conditions, including studies with ATP and ADP under both hydrolytic and non-hydrolytic conditions. In particular we discuss the role of the substrate's intrinsic dynamics for binding of hMsh2-hMsh3 to DNA hairpins and DNA 3-way junctions.