Identification of DNA double strand breaks at chromosome boundaries along the track of particle irradiation

Abstract

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X-ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR-induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon-ion irradiation was >4-fold higher than that after X-ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA-PKcs activity, indicating that the DSB repair is NHEJ-dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon-ion irradiation contain DSBs undergoing DNA-end resection, which promotes repair utilizing microhomology mediated end-joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation. © 2016 Wiley Periodicals, Inc.

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…The existence of more than one DSB under a focus can be an issue for underestimation and the correct assignment of focus size can improve this situation but up to a point. The yields we found for low-LET (Figure 2) are compatible with previous findings of 10-20 DSB foci/Gy/cell [37,42,43] and the same stands also for a-particles [35,44,45] and Ar ions [46], although in the specific study 40 Ar ions of a much lower LET (125 keV/lm) were used. Previous studies using cold lysis PFGE protocols in order to minimize conversion of heat-labile sites to DSBs suggest relatively increased numbers of $25 DSBs/Gy/cell for normal human skin fibroblasts (GM5758) for low-LET radiations [47].…”

“…In general, there is an increase with dose and repair time for both X-rays and 36 Ar ions (steeper increase). These findings reflect the previously introduced idea of radiation-induced foci (RIF) and enlarged repair centers for high-LET radiations following a bimodal response with time, that is increase for 0-24 h and then gradually decrease [10,37].…”

“…This possibility is high especially when the two non-DSB lesions are located in a distance of up to 10 bp [56]. The inaccurate repair of such "hybrid clusters", consisting of DSBs and non-DSBs, can be attributed to the error-prone DNA repair pathways implicated in this case like NHEJ and its backup subpathways alt-EJ (or B-NHEJ) and BER, respectively [7,8,37]. Previous studies by Peddi et al [57] have shown, using an adaptation of PFGE and different types and levels of DNA-PK deficiency, that these deficient cells not only exhibit as expected DSB repair problems, but also the non-DSB oxidative clustered DNA lesions processing is significantly impeded.…”

Clustered DNA double-strand break formation and the repair pathway following heavy-ion irradiation

“…Through the use of immunofluorescence and fluorescence in situ hybridization, we showed that the frequency of γH2AX foci at the chromosome boundary of chromosome 1 following carbon ion irradiation was >4-fold higher than that after X-ray irradiation (Fig. 3A) [29]. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track.…”

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

“…Thus, misrepair resulting in lethal chromosomal rearrangements, such as a dicentirc or a translocation/deletion within an essential gene, can be a critical factor leading to the increased RBE of heavy-ion radiation. We previously demonstrated that heavy-ion particle radiation frequently induces γH2AX foci at chromosome boundaries by using γH2AX-chromosome FISH analysis [ 30 ]. Based on the contact first model, which suggests the joining of two broken chromosomes takes place when the breaks are located in a proximal position [ 31 ], if the clustered DSBs occur at the chromosome boundary, it will likely lead to interchromosomal exchange [ 32 ].…”