Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

Kaufmann, Tanja and Herbert, Sébastien and Hackl, Benjamin and Besold, Johanna Maria and Schramek, Christopher and Gotzmann, Josef and Elsayad, Kareem and Slade, Dea. (2020) Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells. Nucleic Acids Research, 48 (21). e122.

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Official URL: https://edoc.unibas.ch/79640/

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Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Services Biozentrum > Imaging Core Facility (Biehlmaier)
UniBasel Contributors:Herbert, Sébastien
Item Type:Article, refereed
Article Subtype:Research Article
Publisher:Oxford University Press
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:27 Jan 2022 14:56
Deposited On:27 Jan 2022 14:56

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