Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.
AuthorsSobhy, Mohamed Abdelmaboud
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Embargo End Date2019-12-15
Permanent link to this recordhttp://hdl.handle.net/10754/658655
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AbstractBulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule Förster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.
CitationSobhy, M. A., Bralić, A., Raducanu, V.-S., Tehseen, M., Ouyang, Y., Takahashi, M., … Hamdan, S. M. (2019). Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1. Journal of Visualized Experiments, (151). doi:10.3791/60045
SponsorsThis work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H.