Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage
Measurements of protein recruitment and the formation of repair complexes at DNA double-strand breaks in real time provide valuable insight into the regulation of the early DNA damage response. Here, we describe the use of live cell microscopy in combination with ionizing radiation as a tool to evaluate the influence of ATM and its site-specific phosphorylation of target proteins on these processes. Recommendations are made for the preparation of the cells and the design of specialized cell chambers for the localized (and/or targeted) irradiation with charged particles at accelerator beamlines as well as the microscopic equipment and protocol to obtain high-resolution, sensitive fluorescence measurements.
Key wordsDNA damage Double strand breaks (DSB) ATM Live-imaging Protein recruitment GFP Microscopy
This work was supported by BMBF Grants 02NUK037A, 02NUK001A and DFG GRK1657.
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