The presence of DNA repair centers formed by mobilization and clustering of multiple DSBs has been hypothesized by Savage et al and further investigated by our group. In recent years, the use of repair proteins tagged with fluorescent markers has greatly expanded our knowledge of the dynamics and mechanisms of DSB repair. Even a single DSB can kill a cell or even worse lead to genomic instability, which in turn has been shown to contribute to cancer progression. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.ĭouble strand breaks (DSB) are the most deleterious type of DNA damage. The work is made available under the Creative Commons CC0 public domain dedicationĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by the Low Dose Scientific Focus Area, United States Department of Energy.
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This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Received: AugAccepted: Published: June 24, 2015 PLoS ONE 10(6):Įditor: Michael Shing-Yan Huen, The University of Hong Kong, HONG KONG (2015) Characterizing the DNA Damage Response by Cell Tracking Algorithms and Cell Features Classification Using High-Content Time-Lapse Analysis. Such finding may therefore be critical to explain the supralinear dose dependence for chromosomal rearrangement and cell death measured after exposure to ionizing radiation.Ĭitation: Georgescu W, Osseiran A, Rojec M, Liu Y, Bombrun M, Tang J, et al. High doses of ionizing radiation lead to RIF merging into repair domains which in turn increases DSB proximity and misrepair.
We hypothesize that RIF merging reflects a "stressed" DNA repair process that has been taken outside physiological conditions when too many DSB occur at once. This work also highlights DDR which are specific to doses larger than 1 Gy such as rapid 53BP1 protein increase in the nucleus and foci diffusion rates that are significantly faster than for spontaneous foci movement. We estimate repair domain sizes of 7.5 to 11 µm 2 with a maximum number of ~15 domains per MCF10A cell. Large foci formation is shown to be mainly happening through the merging of smaller RIF rather than through growth of an individual focus. We show that DNA repair occurs in a limited number of large domains, within which multiple small RIFs form, merge and/or resolve with random motion following normal diffusion law.
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Using elastic registration techniques based on the nuclear pattern of individual cells, we could describe the motion of individual RIF precisely within the nucleus. High-content-analysis at the single cell level over hundreds of cells allows us to quantify precisely the dose dependence of 53BP1 protein production, RIF nuclear localization and RIF movement after exposure to X-ray. Using automatic extraction of RIF imaging features and linear programming techniques, we were able to characterize detailed RIF kinetics for 24 hours before and 24 hours after exposure to low and high doses of ionizing radiation. Here we report on the dynamics of 53BP1 radiation induced foci (RIF) across multiple cell generations using live cell imaging of non-malignant human mammary epithelial cells (MCF10A) expressing histone H2B-GFP and the DNA repair protein 53BP1-mCherry. However, detailed knowledge of DDR dynamics across multiple cell generations cannot be obtained using a limited number of fixed cell time-points. Traditionally, the kinetics of DNA repair have been estimated using immunocytochemistry by labeling proteins involved in the DNA damage response (DDR) with fluorescent markers in a fixed cell assay.
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