Journal
NUCLEIC ACIDS RESEARCH
Volume 48, Issue 21, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkaa859
Keywords
-
Categories
Funding
- Vienna Science and Technology Fund [LS14-001, NXT17-017]
- Austrian Science Fund [P31112-B28]
- Austrian Academy of Sciences
- City of Vienna
- Austrian Ministry of Science
- Interreg [ATCZ40]
- University of Vienna
- Austrian Science Fund (FWF) [P31112] Funding Source: Austrian Science Fund (FWF)
Ask authors/readers for more resources
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 Forster 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available