Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.
dc.contributor.author | Sobhy, Mohamed Abdelmaboud | |
dc.contributor.author | Bralic, Amer | |
dc.contributor.author | Raducanu, Vlad-Stefan | |
dc.contributor.author | Tehseen, Muhammad | |
dc.contributor.author | Ouyang, Yujing | |
dc.contributor.author | Takahashi, Masateru | |
dc.contributor.author | Rashid, Fahad | |
dc.contributor.author | Zaher, Manal | |
dc.contributor.author | Hamdan, Samir | |
dc.date.accessioned | 2019-11-13T11:37:00Z | |
dc.date.available | 2019-11-13T11:37:00Z | |
dc.date.issued | 2019-10-15 | |
dc.identifier.citation | Sobhy, 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 | |
dc.identifier.doi | 10.3791/60045 | |
dc.identifier.uri | http://hdl.handle.net/10754/660003 | |
dc.description.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. | |
dc.description.sponsorship | This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H. | |
dc.language.iso | en | |
dc.publisher | MyJove Corporation | |
dc.relation.url | https://www.jove.com/video/60045/single-molecule-forster-resonance-energy-transfer-methods-for-real | |
dc.rights | Archived with thanks to Journal of visualized experiments : JoVE | |
dc.subject | Genetics | |
dc.subject | Issue 151 | |
dc.subject | Holliday junction | |
dc.subject | single-molecule FRET | |
dc.subject | homologous recombination | |
dc.subject | 5’ nucleases | |
dc.subject | gap endonuclease I, | |
dc.subject | GEN1 | |
dc.subject | Holliday junction resolvases | |
dc.title | Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1. | |
dc.type | Article | |
dc.contributor.department | Biological and Environmental Sciences and Engineering (BESE) Division | |
dc.contributor.department | Bioscience Program | |
dc.identifier.journal | Journal of Visualized Experiments | |
dc.rights.embargodate | 2021-10-15 | |
dc.eprint.version | Publisher's Version/PDF | |
dc.contributor.affiliation | King Abdullah University of Science and Technology (KAUST) | |
dc.relation.embedded | <iframe allowTransparency="true" allowfullscreen height="415" width="460" border="0" scrolling="no" frameborder="0" marginwheight="0" marginwidth="0" src="https://www.jove.com/embed/player?id=60045&t=1&s=1" ><p><a title="Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1" href="https://www.jove.com/video/60045/single-molecule-forster-resonance-energy-transfer-methods-for-real">Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1</a></p></iframe> | |
pubs.publication-status | Published | |
kaust.person | Sobhy, Mohamed Abdelmaboud | |
kaust.person | Bralic, Amer | |
kaust.person | Raducanu, Vlad-Stefan | |
kaust.person | Tehseen, Muhammad | |
kaust.person | Ouyang, Yujing | |
kaust.person | Takahashi, Masateru | |
kaust.person | Rashid, Fahad | |
kaust.person | Zaher, Manal S. | |
kaust.person | Hamdan, Samir | |
kaust.grant.number | CRG3 | |
refterms.dateFOA | 2019-11-13T11:37:01Z |