Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein–DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.
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We thank C. Joo, S. A. McKinney, I. Rasnik, S. Hohng and S. Myong for experimental help and discussion; C. Murphy, M. Nahas and K. Raghunathan for discussion; T. Ho and A. Niedziela-Majka for help with DNA and protein preparation, respectively; and R. Porter for the SSB expression plasmid. T.H. is an employee of the Howard Hughes Medical Institute. These studies were supported by grants from the National Institutes of Health and the National Science Foundation.
Author Contributions R.R., A.G.K., T.M.L. and T.H. designed the experiments, A.G.K. prepared the wild-type SSB protein and the mutant SSB with fluorescent labels, R.R. performed the experiments and analysed the data; R.R., T.M.L. and T.H. wrote the manuscript.
This file contains Supplementary Material, Supplementary Methods including Table 1, Supplementary Figures 1-12 with legends, and Supplementary References. (PDF 2962 kb)
SSB diffusion movie in three segments. In the first, SSB diffusion via the rolling mechanism is illustrated. In the second, RecA filament growth via monomer addition biases SSB diffusion in a directional manner. In the third, SSB can melt secondary structures transiently via diffusion and promotes RecA filament formation. (MOV 3985 kb)
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Roy, R., Kozlov, A., Lohman, T. et al. SSB protein diffusion on single-stranded DNA stimulates RecA filament formation. Nature 461, 1092–1097 (2009). https://doi.org/10.1038/nature08442
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