Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.

Mohamed Abdelmaboud Sobhy, Amer Bralic, Vlad-Stefan Raducanu, Muhammad Tehseen, Yujing Ouyang, Masateru Takahashi, Fahad Rashid, Manal Zaher, Samir Hamdan

Research output: Contribution to journalArticlepeer-review

Abstract

Bulk 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.
Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number151
DOIs
StatePublished - Oct 15 2019

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledged KAUST grant number(s): CRG3
Acknowledgements: This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H.

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