Imaging DNA damage and cell cycle in living cells
A CFP-53BP1 DNA damage reporter construct designed by Kron Lab. Cyan fluorescent protein is fused to the 53BP1 glycine rich motif (RG), tandem tudor domains for recognition of methylated histones, and a nuclear localization signal (NLS). The construct is expressed under inducible control of the tetracycline-responsive element (TRE).
B Fluorescence Ubiquitination-based Cell Cycle Indicator constructs (FUCCI, Sakaue-Sawano et al.) report red fluorescence in G0/G1, yellow fluorescence at the G1/S transition, and green fluorescence during G2/M cell cycle phase.
C Imaging of live cells expressing reporter constructs conducted by Elena Efimova in Kron Lab. Human MCF-7 breast adenocarcinoma cell lines were exposed to 0 or 6 Gy gamma irradiation and incubated for 24 hours. Numerous DNA damage foci persisted in many cells in response to irradiation.
D Enlarged images of single living cells in discrete cell cycle phases exhibiting persistent DNA damage foci.
E Relationship of cell cycle phase to DNA damage persistence. 53BP1 foci were counted in single cells which were scored as G0/G1 (red), G1/S (yellow), or G2/M (green) based on FUCCI reporter construct emission. Highest mean numbers of persistent DNA damage foci were observed in G0/G1 cells.
- Data contributed by Dr. Elena Efimova, Kron Lab. 2015.