Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

Abstract

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

Keywords: DNA double-strand breaks; alpha particles; high linear energy transfer; homologous recombination; live-cell microscopy; nonhomologous DNA end-joining.

Figure 1

Overview of focus segmentation and focus track analysis. U2OS cells stably expressing p53-binding protein 1 (53BP1)-GFP were irradiated with 2 Gy of α-particle or X-ray irradiation. (A) ImageJ scripts were using for focus segmentation. (B) Center of mass (COM) per focus was calculated. (C) Center of mass was linked to consecutive frames (if COM was ≤ 0.7 µm). Tracks are indicated in red (active track at this time-point) or yellow (past track not active at this time-point). Scale bar indicates 5 µm. Segmentation of foci was correlated to manual counts of foci for both α-particle irradiation (D) and X-ray irradiation (E). For both treatments, three nonconsecutive frames were counted in 10 random nuclei. The averages of these counts are shown.

Figure 2

53BP1-GFP focus resolving after α-particles is slow compared to X-rays. (A) Average number of 53BP1-foci per cell over time. Foci in α-particle-irradiated (2 Gy) cells are depicted in red and those in X-ray-irradiated (2 Gy) cells are depicted in blue. All time-points between the indicated bar are significant differences between X-ray- and α-particle-irradiated cells (ANOVA, p < 0.05). Error bars indicate the standard error of the mean (SEM). (B) Percentage of detected 53BP1-GFP focus tracks that were present in α-particle-irradiated cells (red) or X-ray-irradiated cells (blue) for indicated time. (C–F) Mean square displacement (MSD) curves of the binned track lengths. α-particle-irradiated cells are depicted using a red line (C,D) and X-ray-irradiated cells are shown in blue (E–G). The average apparent diffusion coefficient of 53BP1-GFP foci, which was based on the fit on the first four time intervals.

Figure 3

53BP1-GFP protein accumulation and focus size increase after X-ray irradiation. (A) Average 53BP1-GFP intensity per focus (average pixel intensity) over time after treatment using α-particle (red) or X-ray (blue) irradiation (2 Gy). (B–E) Overview of focus population distribution at 0, 300, 600, and 900 min after the start of imaging. Graphs shown kernel density estimations with the area under the curve being equal to the area of the histogram. The 0 AUs are the consequence of the smoothing procedure used for density estimations. (F) Average 53BP1-GFP focus area trough time. The average of foci induced by α-particles (red) or foci induced by X-ray (blue). (G) Total amount of 53BP1-GFP (product of focus area and pixel intensity) on foci through time after α-particle (red) or X-ray (blue) irradiation. SEM is indicated in gray.

Figure 4

Focus kinetics of 53BP1 and RPA are dose- and radiation type-dependent. Overview of 53BP1 and RPA staining after X-ray (A) and α-particle (B) irradiation using a dose gradient. Cells were fixed 1 h after irradiation. Quantification of 53BP1 foci per EdU-positive cell (C) and intensity (D). Quantification of RPA foci per EdU-positive cell (E) and intensity (F). More than 100 EdU-positive cells were analyzed in two independent experiments. Data points in the plot indicate single nuclei treated with X-ray irradiation (purple) or α-particles (orange). Error bars indicate SEM. Black bars indicate the mean. The statistical differences are indicated by asterisks (***, p < 0.001) and determined by ANOVA followed by Tukey’s multiple comparison test.

Figure 5

Multiple individual resection events after α-particle irradiation. Representative confocal images of induced 53BP1 and RPA foci by X-ray (A.1) or α-particles (A.2). Scale bar indicates 5 µm. (B) Quantification of RPA focus intensity. (C) Percentage of 53BP1 foci colocalizing with 0, 1, or >1 RPA foci. In total, 15 EdU-positive cells were analyzed using structured illumination microscopy for each treatment. (D) Representative images of 53BP1 exclusion at RPA foci after α-particle irradiation. Scale bar indicates 0.8 µm. Per assay, >100 EdU positive cells were analyzed. Error bars indicate SEM. The statistical differences are indicated by asterisks (***, p < 0.001) and determined by student’s t-test.

Figure 6

Working model of the role of radiation type in double-strand break (DSB) repair pathway choice. Both X-ray irradiation and α-particle irradiation induce DSBs. The DNA ends induced by X-ray irradiation are protected by 53BP1 and are repaired by the nonhomologous end-joining (NHEJ) machinery. Repair of DSBs reintroduces 53BP1 back into the general pool and causes the remaining foci to recruit more 53BP1, leading to an increase in focus intensity. Repair via the slow component of NHEJ involves chromatin decondensation, leading to focus growth. On the other hand, after α-particle irradiation, the 53BP1 pool is insufficient, thereby limiting end protection and leading to resection. Resection in the S/G2 phase would activate homologous recombination (HR)-directed repair.