Author:
Subekti Dwiky Rendra Graha,Murata Agato,Itoh Yuji,Takahashi Satoshi,Kamagata Kiyoto
Abstract
AbstractCharacterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the long-lived p53–DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging.
Funder
Research Fellowship for Young Scientists from JSPS
MEXT/JSPS KAKENHI
Publisher
Springer Science and Business Media LLC
Cited by
14 articles.
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