Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

Background: In nuclear medicine, numerous cancer types are treated via internal irradiation with radiopharmaceuticals, including low-LET (linear energy transfer) beta-emitting radionuclides like Lu-177. In most cases, such treatments lead to low-dose exposure of organ systems with β-irradiation, which induces only few isolated DSBs (double-strand breaks) in the nuclei of hit cells, the most threatening DNA damage type. That damaging effect contrasts with the clustering of DNA damage and DSBs in nuclei traversed by high-LET particles (α particles, ions, etc.). Methods: After in-solution β-irradiation for 1 h with Lu-177 leading to an absorbed dose of about 100 mGy, we investigated the spatial nano-organization of chromatin at DSB damage sites, of repair proteins and of heterochromatin marks via single-molecule localization microscopy (SMLM) in PBMCs. For evaluation, mathematical approaches were used (Ripley distance frequency statistics, DBScan clustering, persistent homology and similarity measurements). Results: We analyzed, at the nanoscale, the distribution of the DNA damage response (DDR) proteins γH2AX, 53BP1, MRE11 and pATM in the chromatin regions surrounding a DSB. Furthermore, local changes in spatial H3K9me3 heterochromatin organization were analyzed relative to γH2AX distribution. SMLM measurements of the different fluorescent molecule tags revealed characteristic clustering of the DDR markers around one or two damage foci per PBMC cell nucleus. Ripley distance histograms suggested the concentration of MRE11 molecules inside γH2AX-clusters, while 53BP1 was present throughout the entire γH2AX clusters. Persistent homology comparisons for 53BP1, MRE11 and γH2AX by Jaccard index calculation revealed significant topological similarities for each of these markers. Since the heterochromatin organization of cell nuclei determines the identity of cell nuclei and correlates to genome activity, it also influences DNA repair. Therefore, the histone H3 tri methyl mark H3K9me3 was analyzed for its topology. In contrast to typical results obtained through photon irradiation, where γH2AX and H3K9me3 markers were well separated, the results obtained here also showed a close spatial proximity (“co-localization”) in many cases (minimum distance of markers = marker size), even with the strictest co-localization distance threshold (20 nm) for γH2AX and H3K9me3. The data support the results from the literature where only one DSB induced by low-dose low LET irradiation (<100 mGy) can remain without heterochromatin relaxation for subsequent repair.