Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair

<div><p>The choice of DNA double strand break (DSB) repair pathway is determined at the stage of DSB end resection. Resection was proposed to control the balance between the two major DSB repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Here, we examined the regulation of DSB repair pathway choice at two-ended DSBs following ionizing radiation (IR) in G2 phase of the cell cycle. We found that cells pre-exposed to low-dose IR preferred to undergo HR following challenge IR in G2, whereas NHEJ repair kinetics in G1 were not affected by pre-IR treatment. Consistent with the increase in HR usage, the challenge IR induced Replication protein A (RPA) foci formation and RPA phosphorylation, a marker of resection, were enhanced by pre-IR. However, neither major DNA damage signals nor the status of core NHEJ proteins, which influence the choice of repair pathway, was significantly altered in pre-IR treated cells. Moreover, the increase in usage of HR due to pre-IR exposure was prevented by treatment with ATM inhibitor during the incubation period between pre-IR and challenge IR. Taken together, the results of our study suggest that the ATM-dependent damage response after pre-IR changes the cellular environment, possibly by regulating gene expression or post-transcriptional modifications in a manner that promotes resection.</p></div

BRCA2-depleted cells exhibit an additive repair defect by pre-IR.

<p>A) Pre-IR treatment increases the fraction of BRCA2-knockdown cells with a DNA repair defect. 1BR hTERT cells treated with siControl or siBRCA2 were irradiated with 0.2 Gy X-rays 48 h after siRNA transfection. At 6 h after pre-IR, cells were irradiated with 2 Gy X-rays (challenge IR), and APH was added immediately after the challenge IR. γH2AX foci in G2 cells (CENPF+) were scored. Representative images of G2 cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122582#pone.0122582.s002" target="_blank">S2A Fig</a>. Error bars represent the SD of three independent experiments. B) Knockdown efficiency of BRCA2 is shown. Arrowhead indicates BRCA2. C) Representative images of the increase in the proportion of BRCA2-depleted cells with a DSB repair defect following pre-IR. Scale bar represent 10 μm (for all images).</p

Pre-IR treatment promotes DSB end resection in G2.

<p>A) Schematic diagram of pre-IR treatment. Cells were irradiated with 0.2 Gy X-rays 2 or 6 h before a 2 Gy challenge IR. Cells were fixed and stained for RPA or γH2AX and CENPF 2 h after the challenge IR. APH was added immediately after the challenge IR. B) Pre-IR (0.2 Gy X-rays 6 h prior to challenge IR) promotes IR-induced RPA foci formation. RPA foci in A549 G2 cells (CENPF+) were examined 2 h after 2 Gy X-rays, with or without pre-IR (0.2 Gy X-rays) 2 or 6 h before the challenge IR. (N.B.: although there was a small increase in RPA foci number 2 h after pre-IR, there was no increase in RPA foci 6 h after pre-IR, i.e., the increase in RPA foci following 2 Gy preceded by pre-IR 6 h earlier is not due to damage persisting after the pre-IR treatment.) C) DSB induction is not affected by pre-IR. IR-induced DSB levels after a 2 Gy challenge IR in A549 G2 cells were examined by scoring γH2AX foci 30 min after IR. D) Dose of 0.05 Gy X-rays is sufficient to activate resection. A549 cells were irradiated with 0.05, 0.1, or 0.2 Gy X-rays 6 h prior to a 2 Gy challenge IR. RPA foci in A549 G2 cells were counted 2 h after 2 Gy challenge IR. E) Representative image of the increase in challenge IR-induced RPA foci following pre-IR. RPA foci in A549 G2 (CENPF+) cells are shown 2 h after the 2 Gy challenge IR; cells were exposed to 0.2 Gy pre-IR 6 h previously. Scale bar represent 10 μm (for all images). F) Dose of 0.05–0.2 Gy X-rays does not alter cell-cycle distribution. Cell-cycle distribution in A549 cells was examined by FACS 6 h after pre-IR.</p

Pre-IR treatment promotes the usage of HR, but not NHEJ, in a reporter system.

<p>A) Summary of I-<i>Sce</i>I–based HR and NHEJ assays. HR and NHEJ assays were established as described previously. B) Schematic diagram of pre-IR treatment and I-<i>Sce</i>I induction in the repair assays. C) Pre-IR treatment increases the efficiency of HR. Cells were irradiated with 0.1 or 0.2 Gy X-rays 2 or 6 h prior to I-<i>Sce</i>I transfection, as shown in B. EGFP- or GFP-positive cells were quantitated by FACS 48 h after I-<i>Sce</i>I transfection. Error bars represent the SD of three independent experiments. D) Pre-IR treatment after 12–24 h does not affect HR efficiency. Cells were irradiated with 0.2 Gy X-rays 12 or 24 h prior to I-<i>Sce</i>I transfection. EGFP- or GFP-positive cells were quantitated by FACS 48 h after I-<i>Sce</i>I transfection. Error bars represent the SD of three independent experiments.</p

Analysis of IR-induced DNA damage signaling in cells subjected to pre-IR.

<p>A) The levels of NHEJ proteins did not change in response to pre-IR. A549 cells were harvested 6 h after 0.2 Gy pre-IR. CtIP, which is essential for resection, was also unaffected by pre-IR. B) Histone H3K9me3, which promotes the formation of heterochromatin, was examined in A549 cells 6 h after 0.2 Gy pre-IR. C) ATM signaling, ATM Ser1981 autophosphorylation, and downstream pKAP-1 Ser824 were examined 30 min after 3 Gy (challenge IR) in cells treated with or without 0.2 Gy pre-IR, followed by 6 h incubation.</p

Promotion of resection by pre-IR treatment is ATM-dependent.

<p>A) Schematic diagram of pre-IR treatment with or without ATM inhibitor. A549 cells were irradiated with 0.2 Gy. 10 μM ATM inhibitor was immediately added after 0.2 Gy. After 6 h, fresh medium without ATM inhibitor was added following three washes with PBS. ATM activity recovered immediately following the removal of the inhibitor, as shown in panel C. B) Treatment with ATM inhibitor for 6 h attenuated the increase in RPA foci formation after pre-IR. RPA foci were counted in A549 G2 cells treated with or without 0.2 Gy pre-IR 6 h previously and with or without ATM inhibitor. C) ATM activity recovered following washing after 6 h treatment with ATM inhibitor. Phosphorylation of ATM S1981 and pKAP-1 S824 in A549 cells was examined 30 min after 2 Gy X-rays + 0.2 Gy pre-IR 6 h previously +/- ATM inhibitor treatment. D) Effect of ATM inhibitor was validated by detecting ATM autophosphorylation and KAP-1 phosphorylation. ATM inhibitor was added 15 min before 3 Gy challenge IR. Without removal of ATM inhibitor, A549 cells were harvested 30 min after the challenge IR.</p

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

<div><p>Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.</p></div

Variation in the length and number of tracks between individual flat fibroblast cells.

<p>(A) γH2AX foci in 48BR (WT) primary and 2BN (XLF) hTERT cells were enumerated from 0.5–24 h post 1 Gy X-rays. Foci were scored in 2D using a Zeiss Axioplan microscope. (B) 48BR (WT) cells were fixed at 30 min following 1 Gy Fe ions and stained with γH2AX and DAPI. Asterisks represent non-track cells. (C) Distribution in the percentage of γH2AX tracks following Fe irradiation in B. (D) The average number of tracks per cell following 1 Gy Fe is shown. The predicted fluence of particles traversing the nuclease after 1 Gy horizontal Fe irradiation was estimated to be ∼1.1 with a Poisson distribution predicting and ∼70% cells receiving a particle track (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070107#s2" target="_blank">Materials and Methods</a>). (E) Scatter plots of track length per cell following 1 Gy Fe irradiation are shown in 48BR (WT) and 2BN (XLF) G0/G1 cells. Tracks whose length are <0.025 were excluded from the data, since they were indistinguishable from delta-electron induced foci. ∼250 cells were examined in each analysis (C–E). (F) Clustered γH2AX foci within the tracks is ATM/DNA-PK dependent, indicating that they are DSBs. 48BR (WT) cells were fixed at 30 min post Fe irradiation with/without ATM plus DNA-PK inhibitor. To examine the percentage of track positive cells, >200 cells were scored. Error bars represent the standard deviations (SD) from 2 experiments. The analysis was performed by DeltaVision microscope without deconvolution (B–F). Note that although the cells were exposed to 1 Gy Fe ions, the dose to individual cells can differ due to differing number of particles traversing the cell. In the ensuing analysis, we examine cells which have a single particle traversal.</p

Clustered γH2AX foci arising within the particle tracks represent a signature of high LET particle radiation and are repaired slowly by NHEJ in G1.

<p>(A) 48BR (WT) primary and 2BN (XLF) hTERT G0/G1 cells were irradiated in a horizontal direction with 1 Gy Fe irradiation. Images are taken using the DeltaVision microscope followed by deconvolution. Representative images at 8 and 24 h post Fe irradiation are shown. The nucleus outline is drawn with a dashed line from the DAPI staining. (B, C) Percentage of individual foci within a cluster was analysed from >100 individual clusters at each time point. Similar results were obtained in two independent experiments. Cells forming a single γH2AX track of length >8 µm and width >1 µm were analysed.</p

Track structure simulation and spatial distribution of DSBs arising from an Fe ion track.

<p>A Simulation of the structure of damage arising from a single Fe ion particle traversal. The black marks and the red marks represent the tracks of Fe ions and the position of DSBs, respectively. The blue ellipsoid shape shows the size of the nucleus. The slighter wider band of the damage within the tracks observed in these experiments is likely explained by DNA movement after radiation exposure. The number of electrons generated decreases as the electron energy decreases. For 416 MeV/n Fe ions, ∼90% of electrons generated are estimated to have energies less than 100 eV. The range of such low-energy electrons is less than a few nm. The majority of other electrons have a range from a nm to 5 µm. The highest energy electrons with a range of a few hundred µm can also arise but at a low frequency (<0.1%).</p