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Molecular Biology

Live cell-imaging of DNA repair: DNA doublestrand breaks (DSBs) induced by ionizing irradiation (IR)

In an interdisciplinary collaboration between the Physikalisch-Technische Bundesanstalt (PTB) and the DSMZ, live-cell imaging has been established at the charged-particle microbeam facility of PTB. An improved assessment of the biological effects and related risks of low doses of IR is currently an important issue in radiation biology.

Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterize and use highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions.

Foci-formation kinetics in HT-1080 cells showed a delay of about 20 s for p53BP1 foci detection compared to wild-type MDC1, confirming the hierarchical assembly of both proteins for high-LET radiation (Figure 1). Preliminary data for proton irradiations showed no differences in the order of binding proteins. Efforts to improve the quantity and quality of the data for irradiations with protons, and consequently the reliability of the analysis, are presently undertaken (1).

Figure 1: Cells of the fibrosarcoma cell line HT-1080 stably expressing p53BP1 were irradiated with α-particles of LET of 75 keV/µm. Foci formation could be detected in a reliable manner by both fusion proteins, (data for MDC1 not shown). Co-localization of γ-H2AX foci confirmes the DSB nature of the detected radiation-induced fluorescence foci.
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Dr. Dirks, Wilhelm
Phone: +49-531/2616-166

Molecular Biology

Reference:

  1. Giesen U, Langner F, Mielke C, Mosconi M, Dirks WG. Online imaging of initial DNA damages at the PTB microbeam. Radiat Prot Dosimetry 143: 349-352 (2011).