Probing Protein Diffusion and Dissociation Mechanisms on DNA Using Fluorescence-Force Spectroscopy

Abstract

Single-stranded (ss)DNA binding (SSB) proteins tightly bind to ssDNA and protect it from degradation during DNA replication, recombination and repair. For subsequent DNA processing, SSB proteins need to be displaced from ssDNA and replaced by other proteins. The recently discovered activity that E. coli SSB can diffuse on ssDNA [1] may facilitate these processes, but little is known about the diffusion mechanism. Here we use single-molecule fluorescence-force spectroscopy [2] to study DNA-protein interactions and show that ssDNA can be progressively unraveled from the surface of a single E. coli SSB tetramer with increasing force between 1-6 pN, followed by SSB dissociation at about 9 pN. Our data also indicate that SSB diffuses on ssDNA primarily via a reptation rather than a rolling mechanism. These approaches provide unique insights into the mechanical regulation of DNA-SSB interactions and are generally applicable to many other protein-nucleic acid systems.[1] R. Roy, Kozlov, A. G., Lohman, T. M. and T. Ha. SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature (2009, in press)[2] S. Hohng, R. Zhou, M. K. Nahas, J. Yu, K. Schulten, D. M. J. Lilley and T. Ha. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318, 279-283 (2007).