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dc.contributor.authorSobhy, Mohamed Abdelmaboud
dc.contributor.authorBralic, Amer
dc.contributor.authorRaducanu, Vlad-Stefan
dc.contributor.authorTehseen, Muhammad
dc.contributor.authorOuyang, Yujing
dc.contributor.authorTakahashi, Masateru
dc.contributor.authorRashid, Fahad
dc.contributor.authorZaher, Manal
dc.contributor.authorHamdan, Samir
dc.date.accessioned2019-11-13T11:37:00Z
dc.date.available2019-11-13T11:37:00Z
dc.date.issued2019-10-15
dc.identifier.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
dc.identifier.doi10.3791/60045
dc.identifier.urihttp://hdl.handle.net/10754/660003
dc.description.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.
dc.description.sponsorshipThis 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.isoen
dc.publisherMyJove Corporation
dc.relation.urlhttps://www.jove.com/video/60045/single-molecule-forster-resonance-energy-transfer-methods-for-real
dc.rightsArchived with thanks to Journal of visualized experiments : JoVE
dc.subjectGenetics
dc.subjectIssue 151
dc.subjectHolliday junction
dc.subjectsingle-molecule FRET
dc.subjecthomologous recombination
dc.subject5’ nucleases
dc.subjectgap endonuclease I,
dc.subjectGEN1
dc.subjectHolliday junction resolvases
dc.titleSingle-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1.
dc.typeArticle
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.contributor.departmentBioscience Program
dc.identifier.journalJournal of Visualized Experiments
dc.rights.embargodate2021-10-15
dc.eprint.versionPublisher's Version/PDF
dc.contributor.affiliationKing 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-statusPublished
kaust.personSobhy, Mohamed Abdelmaboud
kaust.personBralic, Amer
kaust.personRaducanu, Vlad-Stefan
kaust.personTehseen, Muhammad
kaust.personOuyang, Yujing
kaust.personTakahashi, Masateru
kaust.personRashid, Fahad
kaust.personZaher, Manal S.
kaust.personHamdan, Samir
kaust.grant.numberCRG3
refterms.dateFOA2019-11-13T11:37:01Z


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