High-LET targeted microbeam irradiation induces local chromatin reorganization in living cells showing active basal mechanisms at highly complex DNA damage sites

Abstract

DNA repair eukaryotic cells have additional protective mechanisms that avoid uncontrolled interaction of different parts of the chromatin and damaged regions. Key factors here are the regulation of chromatin density and mobility. The 4D (temporal and spatial) organization of chromatin is controlling this security barrier by regulating the accessibility of genes, flexibility of DNA, and its ability to move inside the nucleus. How this regulation mechanisms are involved in DNA repair upon radiation damage is until now rarely known but an important part to understand the enhanced effectiveness of high linear energy transfer (LET) particles. The damage recognition via PARP1 and the subsequent chromatin decondensation via PARylation is a crucial step in the DNA damage response (DDR). Upon We used the SNAKE microbeam with a beam spot size of <1 µm to induce highly localized DNA damage in living cells using 55 MeV Carbon ions to investigate the chromatin rearrangements in the early stage of DDR. The nuclei were irradiated with a cross pattern consisting of 1000 ions per spot and 25 spots per cell either with one (11 000 Gy), two (22 000 Gy), or three crosses (33 000 Gy). The chromatin rearrangement was imaged live for several minutes after irradiation at the beam using SiR chromatin stain. Upon 91% of the cells show a localized decondensation starting from a few seconds up to minutes after irradiation. The chromatin is decondensed by 6%-8% in the beam path with a local condensation at the edges of up to 8%. Our results suggest that chromatin decondensation is a fast process in the first few seconds after damage induction. Furthermore, decondensation status does not change over minutes, which gives evidence that this process and therefore DDR is paused or even stopped. In combination with the existing knowledge about early reactions to damage induction our data support the model of PARP induced chromatin decondensation. Furthermore, it is evident that also ultra-high doses of radiation are, in first place not able to inactivate initial basal mechanisms as response to damage induction.