Distribution and dynamics of charged particle-induced DNA double-strand breaks

• Main Author: Splinter, Jörn
• Format: Others
• Language: English; en
• Published: 2010
• Online Access: https://tuprints.ulb.tu-darmstadt.de/2228/2/Dissertation.pdf; Splinter, Jörn <http://tuprints.ulb.tu-darmstadt.de/view/person/Splinter=3AJ=F6rn=3A=3A.html> (2010): Distribution and dynamics of charged particle-induced DNA double-strand breaks.Darmstadt, Technische Universität, [Ph.D. Thesis]

The efficient repair of DNA double-strand breaks (DSBs) is clearly decisive in determining the ‘fate’ of damaged cells, but the spatiotemporal organisation of repair events that might explain the formation of chromosomal misrejoining and genome instability is not yet clear. Following generation of DSBs the histone variant H2AX is phosphorylated (gH2AX) comprising megabase-pair regions of the chromatin around the DSB (Rogakou et al. 1998; Rogakou et al. 1999) that can be visualized by immunostaining. Irradiation of cell nuclei with charged particles leads to the spatially defined production of DSBs along the particle trajectory, thus facilitating studies on the dynamics of ionizing radiation-induced foci (IRIF) associated with lesion processing. Microscopic imaging of ion-induced IRIF patterns in fixed and living cells revealed that lesion density has only a minor impact on the pattern and number of gH2AX IRIF (Chapter Four) and showed a general positional stability of DNA lesions (Chapter Five), respectively. The former finding demonstrates that the number of visualized IRIF following ion irradiation is below the amount expected for the applied doses and it was suggested that single IRIF might contain several damage sites corresponding to the high lesion density induced by ions (Jakob et al. 2003; Costes et al. 2007). We addressed this question using repair-related proteins forming smaller (micro-)foci compared to gH2AX, but despite some gH2AX IRIF containing multiple micro-foci its number was still lower than expected. Therefore, high-resolution 4Pi microscopy was applied (AG Hell, DKFZ Heidelberg) to resolve a potential substructure of micro-foci. However, a substructure was only observed for gH2AX and 53BP1 signals, but not for the micro-foci forming repair-proteins RPA and hMre11 (Chapter Six). Nevertheless, despite the restricted dynamic range of foci numbers following ion irradiation of different LETs the application of micro-foci marker allowed a rough estimation of the dose deposited by UVA laser microirradiation. For this laser-induced RPA IRIF patterns were compared with patterns induced by low angle ion irradiation (Jakob et al. 2003) of different LETs (Chapter Six). The laser dose was estimated to be in the range of hundreds of Gray. As a further aspect of IRIF patterns an influence of chromatin structure on the foci positions was recently discussed by Costes et al. (2007) reporting a preferential localization of gH2AX signals at the interface between regions of low and high intensive DNA staining. In agreement with the here described slow and confined damage motion (Chapter Five), these authors hypothesized that a potential small range damage translocation might occur. To elucidate such a dynamic process we used low angle and targeted single ion irradiation to induce DSBs spatially confined inside heterochromatic compartments in mammalian cells and analyzed the 3D geometry of induced gH2AX and XRCC1 signals at different time point post-irradiation (Chapter Seven). We demonstrate that, contrary to the current notion, phosphorylation of H2AX is indeed possible within heterochromatin and that damage sites induced in the interior of heterochromatic compartments are expelled to the periphery within 20 min. We further show that this relocation is independent of ATM, a protein previously reported to be involved in the repair of heterochromatin-associated damages (Goodarzi et al. 2008). Taken together, the here described results suggest that chromatin structure and not an repair-related directional mobility of damage sites is responsible for the preferentially localization of IRIF at the border of intensively stained chromatin regions and for the characteristic gap structure of ion-induced IRIF patterns.