Measuring Search Times in Site Specific DNA Binding

Abstract

Facilitated diffusion is the accepted theory of how site-specific DNA binding proteins search for their targets. When DNA sliding is included in the search strategy, protein search times are predicted to exhibit a bi-model dependence on the sliding length, with a minimum occurring at an optimal sliding length. This peak in specific association rate as sliding length is varied has rarely been directly measured. We are studying facilitated diffusion using restriction endonucleases as a model system. Our multiplexing single molecule technique is based on DNA tethering of microbeads and can measure the time of cleavage of up to hundreds of DNAs in a single experiment. We use diffusion controlled conditions to directly measure the search time of proteins which cleave dsDNA. In the work reported here, we vary the siding length by varying the salt concentration from a few mM up to 150 mM NaCl. We have discovered a peak in specific association rates of both EcoRI and NdeI, consistent with the predictions of facilitated diffusion. These peaks both occur near 80 mM NaCl, significantly less than physiological salt concentrations. I will discuss how our technique works and how we are using the theory of facilitated diffusion to understand our results. I will further discuss our efforts to directly verify DNA sliding in these systems using single molecule fluorescent imaging.