Abstract
We have developed a new high-throughput single-molecule technology that allows us to monitor hundreds of individual DNA molecules that are aligned in parallel arrays on the surface of a microfluidic sample chamber that is coated with a fluid lipid bilayer. This method relies on the use of hydrodynamic force to organize lipid-tethered DNA molecules along the leading edge of a mechanical barrier to lipid diffusion. Using total internal reflection fluorescence microscopy (TIRFM), we can directly visualize the behavior of thousands of individual protein complexes in real time as they interact with the DNA molecules within the parallel array. We are using this technology to study the biochemical mechanisms of DNA repair. Here we demonstrate that the human DNA repair protein Rad51 can assemble into rings on double and these rings can slide long distances on the DNA via a one-dimensional diffusion mechanism. Surprisingly, the proteins stop sliding and remain tightly bound when they encounter the “broken” end of a linear DNA molecule. This suggests that 1D-diffusion may play a role in the assembly of protein complexes during DNA damage repair. This research was supported in part by Startup funds from Columbia University, the Susan G. Komen Foundation, and the Irma T. Hirschl and Monique Weill-Caulier Trusts.
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