Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer |
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| Authors: | Kyung Suk Lee Amanda B Marciel Alexander G Kozlov Charles M Schroeder Timothy M Lohman Taekjip Ha |
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| Institution: | 1 Department of Physics, Center for Physics in Living Cells and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801–2902, USA;2 Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, IL 61801, USA;3 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110–1093, USA;4 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, IL 61801, USA;5 Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801–2902, USA |
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| Abstract: | Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt2/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation. |
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| Keywords: | SSB single-stranded DNA binding protein ssDNA single-stranded DNA RCR rolling circle replication TIRF total internal reflection fluorescence MSD mean square displacement dsDNA double-stranded DNA |
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