Abstract
In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.
Highlights
The DNA organized into the chromatin in the eukaryotic cell nucleus is permanently attacked and damaged by environmental factors such as chemicals and drugs [1,2,3,4] or, for instance, UV or ionizing radiation (e.g., X-rays, particles of atomic decays, etc.) [5,6]
In the context of double strand breaks (DSBs) repair, several repair pathways can be distinguished based on the requirement for the DNA-end resection and presence of homologous repair templates: (a) non-homologous end-joining (NHEJ) [17,18,22,23,24], the fast and seemingly most often used pathway in mammals; (b) homologous recombination (HR) [19,23,25,26,27], the error-free but slower pathway; and (c) alternative or back-up end-joining mechanisms (a-Ej) [28,29,30,31,32], whose classification is not yet entirely obvious as they combine aspects of both NHEJ and HR to varying degrees
The trick for microscopy is to separate the signals of individual fluorochromes in time and in space [67,74] (Figure 1A,B)
Summary
The DNA organized into the chromatin in the eukaryotic cell nucleus is permanently attacked and damaged by environmental factors such as chemicals and drugs [1,2,3,4] or, for instance, UV or ionizing radiation (e.g., X-rays, particles of atomic decays, etc.) [5,6]. Such damage may dramatically impact intracellular processes such as energy metabolism, DNA replication or protein synthesis (see, for example, [4,7,8,9,10]). Single-chain hybridization (single-strand annealing, SSA) [31] and microhomology-mediated end-joining (MMEJ) [33,34] can be mentioned as commonly appearing as A-Ej in the literature
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