Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair

•TEM permits nanoscale visualisation of DSBs and clustered lesions in chromatin.•Low-LET radiation induces isolated, readily repairable DSBs in entire nuclei.•High-LET radiation induces multiple, closely spaced DSBs in particle tracks.•Highly clustered lesions persist unrepaired in compact heterochromatin.•Nanoscale resolution of TEM revealed higher DSB yields, particularly in tracks. Low- and high-linear energy transfer (LET) ionising radiation are effective cancer therapies, but produce structurally different forms of DNA damage. Isolated DNA damage is repaired efficiently; however, clustered lesions may be more difficult to repair, and are considered as significant biological endpoints. We investigated the formation and repair of DNA double-strand breaks (DSBs) and clustered lesions in human fibroblasts after exposure to sparsely (low-LET; delivered by photons) and densely (high-LET; delivered by carbon ions) ionising radiation. DNA repair factors (pKu70, 53BP1, γH2AX, and pXRCC1) were detected using immunogold-labelling and electron microscopy, and spatiotemporal DNA damage patterns were analysed within the nuclear ultrastructure at the nanoscale level. By labelling activated Ku-heterodimers (pKu70) the number of DSBs was determined in electron-lucent euchromatin and electron-dense heterochromatin. Directly after low-LET exposure (5min post-irradiation), single pKu70 dimers, which reflect isolated DSBs, were randomly distributed throughout the entire nucleus with a linear dose correlation up to 30Gy. Most euchromatic DSBs were sensed and repaired within 40min, whereas heterochromatic DSBs were processed with slower kinetics. Essentially all DNA lesions induced by low-LET irradiation were efficiently rejoined within 24h post-irradiation. High-LET irradiation caused localised energy deposition within the particle tracks, and generated highly clustered DNA lesions with multiple DSBs in close proximity. The dimensions of these clustered lesions along the particle trajectories depended on the chromatin packing density, with huge DSB clusters predominantly localised in condensed heterochromatin. High-LET irradiation-induced clearly higher DSB yields than low-LET irradiation, with up to ∼500 DSBs per μm3 track volume, and large fractions of these heterochromatic DSBs remained unrepaired. Hence, the spacing and quantity of DSBs in clustered lesions influence DNA repair efficiency, and may determine the radiobiological outcome.