3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation

Abstract

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

High linear energy transfer carbon-ion irradiation increases the release of the immune mediator high mobility group box 1 from human cancer cells

“…A limitation of the present study is the lack of proof of the relationship between levels of HMGB1 release and number of DSBs in tumor cells. By quantification of clustered DSBs using a super-high-resolution microscope system, Hagiwara et al suggested that closely localized DSBs are a risk factor for the formation of chromosomal rearrangement after C-ion RT [ 26 ]. The use of such a high-resolution microscope system may facilitate understanding of HMGB1 release under high-LET radiation.…”

Murine polyomavirus DNA transitions through spatially distinct nuclear replication subdomains during infection

“…In cells undergoing DNA damage repair, RPA forms foci that are similar to those observed in VRCs [49,50]. To determine if the focal RPA32 was associated with DDR signaling, the hyperphosphorylated form of RPA32 (pRPA32 S4S8 ) and phosphorylated ATM kinase (pATM S1981 ) were visualized.…”

A Simplified Cluster Analysis of Electron Track Structure for Estimating Complex DNA Damage Yields

“…Experimental methods for detecting complex DNA damage have evolved in recent decades [24,[30][31][32][33][34][35][36]. Among several techniques [24,[30][31][32][33][34][35][36], microscopic operations coupled with an antibody against γ-H2AX [31][32][33][34] enables researchers to obtain spatial distributions of DSB induction in the cell nucleus. The adjacent degree of the DSB site can be evaluated from γ-H2AX foci volume in an assay [35,36]; however, the damage complexity at the nano-meter scale (the scale of DNA) cannot be obtained due to the limited spatial resolution from hundreds of nm to a few µm [35,36].…”

“…Among several techniques [24,[30][31][32][33][34][35][36], microscopic operations coupled with an antibody against γ-H2AX [31][32][33][34] enables researchers to obtain spatial distributions of DSB induction in the cell nucleus. The adjacent degree of the DSB site can be evaluated from γ-H2AX foci volume in an assay [35,36]; however, the damage complexity at the nano-meter scale (the scale of DNA) cannot be obtained due to the limited spatial resolution from hundreds of nm to a few µm [35,36]. Meanwhile, complex DNA damage composed of BD can be quantified by means of gel electrophoresis after treatment of base excision repair enzymes [24,30,37,38] and fluorescence resonance energy transfer [39,40].…”