Loading Dynamics of a Sliding DNA Clamp |
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Authors: | Dr Won‐Ki Cho Dr Slobodan Jergic Daehyung Kim Prof Nicholas E Dixon Prof Jong‐Bong Lee |
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Institution: | 1. Department of Physics, School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang (Korea);2. Centre for Medical and Molecular Bioscience, School of Chemistry, University of Wollongong, Wollongong, N.S.W. 2522 (Australia) |
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Abstract: | Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E. coli β clamp in real time by using single‐molecule FRET (smFRET). Three successive FRET states were retained for 0.3 s, 0.7 s, and 9 min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3 s, and after 0.7 s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9 min. An additional single‐molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single‐molecule polarization and FRET studies thus revealed the real‐time dynamics of the ATP‐hydrolysis‐dependent 3D conformational change of the β clamp during loading at a ss/dsDNA junction. |
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Keywords: | DNA clamps DNA replication single‐molecule FRET single‐molecule polarization ternary complexes |
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