Spatio-temporal dynamics of damage response proteins: the impact of lesion and chromatin complexity
The use of charged particles in radiotherapy has been increasing due to the advantageous tumor dose conformation and - for heavier ions such as carbon – due to their increased biological efficacy. Moreover, in view of their spatially localized dose deposition, further applications of ion beams in therapy are conceivable. At the molecular level, heavy ions deposit large amounts of energy along the ion trajectories, leading to the induction of highly localized DNA lesions including clustered double-strand breaks (DSBs) that pose a challenge to cellular repair.
Clustered DSBs are indeed slower and less efficiently repaired, with complex DSBs associated to heterochromatin (HC) being stronger impaired compared to euchromatin. Using aimed ion irradiation to produce localized damage centrally within murine HC compartments, we found that DSBs are subsequently relocalized to the HC periphery for repair. Moreover, the increasing complexity of DSBs promotes the resection of DNA ends in all cell cycle phases, most likely committing to slow and error prone repair pathways in G1 cells. Therefore, we study the regulation of DSB resection upon induction of complex lesions in this cell-cycle phase using replication protein A (RPA) as a resection marker. We find that the factor CtIP and ubiquitination via RNF138 are important for resection in G1 cells.
The heavy ion-induced damage tracks are used in live cell microscopy studies to assess the spatiotemporal dynamics of DNA repair proteins and the influence of lesion complexity. The measurement of DNA DSB motion in mammalian cell nuclei revealed a remarkable positional stability independent of lesion complexity. Furthermore, based on the track structure of high-energetic particles we simultaneously produced complex and simple DSBs in the same nucleus. We observed that simple DSBs generated in the penumbra region behaved similar to X-ray-induced lesions, showing less persistent foci and an unexpected temporal variability in the sensing of individual DSBs within single nuclei.
Fluorescence-Lifetime Imaging Microscopy (FLIM) yields additional information on changes in the environment like chromatin decompaction. In combination with multi-photon excitation FLIM will allow the label free probing of radiation-induced changes of the redox status of single cells by sensing NAD(P)H. Overall, the combination of particle irradiation with various live cell microscopy approaches will contribute to a more comprehensive picture of the cellular response to radiation.
This work is partially funded by the BMBF grant 02NUK037A and part of the DFG graduate school GRK1657
HBiophysics, GSI Helmholtzzentrum für Schwerionenforschung, Darmstadt, Germany
Domain 1 - UMR 3347 / U1021 - Normal and pathological signaling