Direct measurement of protein-protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells.
Nucleic Acids Res
; 48(21): e122, 2020 12 02.
Article
em En
| MEDLINE
| ID: mdl-33053171
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Osteoblastos
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Fatores de Transcrição
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Mapeamento de Interação de Proteínas
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Poli(ADP-Ribose) Polimerase-1
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Proteína 1 Complementadora Cruzada de Reparo de Raio-X
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article