2. First Insights into DSB Repair and its Regulation at the Nanoscale

Microscopic research of DSB repair is further complicated by the fact that some proteins of which only a few molecules are needed, do not form extended, microscopically distinguishable ionizing radiation induced foci (IRIFs) and can thus be visualized only by superresolution visualization methods. Electron microscopy has yielded surprising results regarding the focal accumulation of repair proteins in euchromatin and heterochromatin. Lorat et al. [52,103], who analyzed the nuclear distribution of various repair proteins in cells irradiated with low-LET and high-LET radiation showed that at two time periods post irradiation (0.5 and 5 h), γH2AX, MDC1, and 53BP1 can be detected only in heterochromatin domains positive for H3K9me3, while the Ku70/80 heterodimer can be detected in both euchromatin and heterochromatin. This observation strongly suggests the involvement of the micro- and/or nanoarchitecture of chromatin and, subsequently, IRIFs in the selection and/or propagation of a particular repair pathway. However, the absence of the indicated protein foci in euchromatin has not been confirmed by any other technique and contradicts the results of confocal microscopy.

Methodologically, this contradiction may be explained by a gap in resolution and thus a gap in knowledge in the 50 nm to 200 nm scale range. Whereas the strength of electron microscopy lies in the low-10-nm scale range, optical confocal microscopy covers resolutions above 200 nm. Thus, for decades during the second half of the 20th century, the scale range between electron and confocal microscopy, although highly relevant for biomolecular dynamics, seemed to be obscured for gaining scientific insights. Therefore, great hopes are currently placed in emerging studies using superresolution light microscopic techniques [134,135], which cover this critical gap of visualization and approach a resolution of 10 nm while preserving the advantages of optical microscopy.

We recently introduced single-molecule localization microscopy (SMLM) [136,137] to simultaneously analyze the architecture of damaged chromatin domains and IRIFs at the nanoscale (see, for example, Figure 6; compare the widefield image, panel A, with SMLM images, panels B–D) [106,138,139]. SMLM is one of the superresolution (nanoscopy) techniques established in recent decades [140]. In addition to having an improved resolution of approximately 10 nm, SMLM is renowned for providing quantitative data on 2D/3D localization (coordinates) and other signal parameters of individual molecules of interest without a need for complicated image analysis. Several SMLM and other nanoscale studies have shown that IRIFs have an internal nanoarchitecture, with nanoclusters of γH2AX and individual proteins occupying nonoverlapping space [141,142].

With the SMLM data matrix of molecule coordinates, Ripley’s metrics for pairwise distance frequency histograms [139,143] can be applied to evaluate structures, molecular clusters, or spatial distributions of label points and their dynamic rearrangements during repair (Figure 7; compare panels A and B for 500 mGy and 4 Gy exposure to X rays) [106,141,144]. Using these approaches of structure elucidation from distance frequency patterns, together with newly developed mathematical topological tools based on persistent homology [145], we showed (for SkBr3 cells exposed to 1 Gy X-rays) that the topological similarity and, thus, the nanoarchitecture of γH2AX clusters depends on the distance of the clusters from heterochromatin (Figure 6) [106]. High topological similarities were also found for 53BP1 clusters in repair foci along high-LET ¹⁵N particle tracks in neonatal human dermal fibroblasts (NHDF) and the U87 glioblastoma cell line [120]. More generally, this finding means that the architecture of γH2AX and 53BP1 clusters is not random and depends on the chromatin environment at DSB sites, consistent with the results of high-resolution ChIP-seq mapping of γH2AX spreading from multiple DSBs induced at annotated positions in human DIvA cells [146]. This finding shows that phosphorylation follows a highly stereotyped pattern governed by the original (predamage) chromatin architecture. Provided that the chromatin architecture dictates γH2AX spreading, it is reasonable to suppose that the architecture of nascent γH2AX foci subsequently affects downstream repair events. Such events could be the binding and organization of repair proteins (such as MDC1 and 53BP1) to IRIFs, the insertion of epigenetic marks (e.g., ubiquitin) into IRIFs, and, in turn, the determination of the architecture of maturating or already dissolving IRIFs [147].

Indeed, the roles of numerous proteins in DSB repair dramatically depend on the specific conditions. The 53BP1 protein generally inhibits resection and promotes NHEJ [148]. However, at some DSB substrates, it shows the opposite effects. For instance, it stimulates resection and switches NHEJ to MMEJ [8,11,149]. In addition, 53BP1 enhances repair fidelity independent of the repair pathway [150]. Hence, 53BP1 and some other proteins, such as BRCA1, probably establish structural platforms that support the recruitment and assembly of the repair machinery in specific ways, dictated by integrated information from multiple global and local factors (reviewed in [151]). Strikingly, 53BP1 and RIF1 were only recently discovered to form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage [150].

Our SMLM analysis also revealed that the nanoarchitecture of γH2AX foci in heterochromatin shows a higher mutual similarity than γH2AX foci in euchromatin. These greater differences between IRIFs in euchromatin probably reflect the variability in the expression intensity across euchromatin loci, in contrast to the rather uniformly silenced heterochromatin. On the other hand, heterochromatin experiences especially extensive architectural reorganization associated with repair initiation and progression. Thus, γH2AX foci in euchromatin may still reflect the variable original architectures of differently expressed genomic domains, but the architecture of γH2AX foci in heterochromatin has already adopted the features of remodeling. Our results thus suggest that remodeling processes at different sites in heterochromatin broadly follow the same principles, indicating that the same repair mechanism is active across these sites. This situation is in contrast to the variable repair of DSBs in structurally and functionally heterogeneous euchromatin.

In addition, using SMLM, we showed that the formation kinetics and architecture of 53BP1 foci differ for normal (nontransformed) and tumor cells, represented in the study by human dermal fibroblasts and highly radioresistant U87 glioblastoma cells, respectively [120]. The data currently being processed seem to suggest that γH2AX, RAD51, MRE11 and potentially other repair proteins also form IRIFs with cell type-specific kinetics and architecture. These differences might contribute to differences between the cells in repair pathway utilization and capacity.

Other breakthrough studies supporting the idea that IRIF nanoarchitecture reflects the repair mechanism or even significantly contributes to the repair pathway choice were published by Reindl et al. [121,152]. By using STimulated Emission Depletion (STED) microscopy [153], another well-established superresolution fluorescence microscopy technique, the authors showed that two nanoscale subzones exist within HR- but not NHEJ-associated IRIFs. Specifically, the resection zone and the zone of surrounding modified chromatin were recognized in [121,152] and in our preliminary unpublished analyses. Furthermore, IRIFs formed by different repair proteins that have specific functions in the NHEJ and HR pathways, such as 53BP1, BRCA1 and RAD51, were clearly shown to have different architectures ([121,152] and our unpublished results). Finally, mutual reorganization of 53BP1, BRCA1 and RAD51 proteins in the frame of IRIFs correlated with switching between NHEJ and HR [121,152]. Hence, the HR, NHEJ, and perhaps A-Ej pathways seem to form IRIFs with characteristic architectures.

Several other studies on IRIF nanoarchitecture have recently been published but cannot be discussed here due to space limitations. However, these studies focus on IRIF ultrastructure rather than the relationship of this ultrastructure to the selection of repair pathways [154]. Future experiments on synchronized cells, cells with altered/manipulated DSB repair pathways, or cells exposed to high-LET ions, as discussed below, are expected to provide more accurate insights into the relationship between the repair mechanisms and nanoarchitecture of particular chromatin domain types and IRIFs.