TY - JOUR
T1 - Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.
AU - Sobhy, Mohamed Abdelmaboud
AU - Bralic, Amer
AU - Raducanu, Vlad-Stefan
AU - Tehseen, Muhammad
AU - Ouyang, Yujing
AU - Takahashi, Masateru
AU - Rashid, Fahad
AU - Zaher, Manal
AU - Hamdan, Samir
N1 - 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.
PY - 2019/10/15
Y1 - 2019/10/15
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/660003
UR - https://www.jove.com/video/60045/single-molecule-forster-resonance-energy-transfer-methods-for-real
UR - http://www.scopus.com/inward/record.url?scp=85073144992&partnerID=8YFLogxK
U2 - 10.3791/60045
DO - 10.3791/60045
M3 - Article
C2 - 31609352
SN - 1940-087X
JO - Journal of visualized experiments : JoVE
JF - Journal of visualized experiments : JoVE
IS - 151
ER -