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A rapid mixing-photocrosslinking technique to study the dynamics of nucleic acid-protein interactions

✍ Scribed by Cheng-Wen Wu; Zaharia Hillel; Chan Suk Park

Publisher
Elsevier Science
Year
1983
Tongue
English
Weight
794 KB
Volume
128
Category
Article
ISSN
0003-2697

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✦ Synopsis

A rapid mixing-photocrosslinking technique has been developed to investigate the kinetics of protein-nucleic acid interactions. With this technique, binding of nucleic acid to protein is first synchronized by rapid mixing in a stopped-flow apparatus. The intermediates formed at different stages of the binding process are then "frozen" by photocrosslinking with a 10-microseconds uv light pulse at various times after mixing. By analyzing structural changes of these intermediates as a function of time, one can obtain the information concerning the dynamic aspects of the interaction. This technique may also be applied to other macromolecular interactions in biological systems.

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Fluorescamine is a useful reagent in monitoring protein-DNA interactions only if a convenient method of separating the complex from free protein is available. Sedimentation of the complex provides such a method at least in the case of histone-like proteins capable of extensive interaction with DNA.