Fig 1.
Pre-IR treatment promotes the usage of HR, but not NHEJ, in a reporter system.
A) Summary of I-SceI–based HR and NHEJ assays. HR and NHEJ assays were established as described previously. B) Schematic diagram of pre-IR treatment and I-SceI 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-SceI transfection, as shown in B. EGFP- or GFP-positive cells were quantitated by FACS 48 h after I-SceI 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-SceI transfection. EGFP- or GFP-positive cells were quantitated by FACS 48 h after I-SceI transfection. Error bars represent the SD of three independent experiments.
Fig 2.
BRCA2-depleted cells exhibit an additive repair defect by pre-IR.
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 S2A Fig. 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).
Fig 3.
Pre-IR treatment promotes DSB end resection in G2.
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.
Fig 4.
Pre-IR promotes resection-dependent RPA phosphorylation at S4/8.
A) RPA Ser4/8 phosphorylation, which is correlated with levels of resection, is also promoted by pre-IR treatment. A549 cells were harvested 2 and 4 h after a challenge IR of 20 Gy; 6 h previously, cells were exposed to 0.2 Gy pre-IR (N.B.: due to limits on assay sensitivity, high-dose IR is required to detect RPA Ser4/8). The level of the MRE11–NBS1–RAD50 complex, which plays a central role in resection, was not altered by pre-IR treatment. Ku80, which is a NHEJ factor, was used as a loading control. B) pRPA levels were quantitated using ImageJ. The quantitation was performed on a single blot shown in panel A. Similar results were obtained in more than two independent experiments. Because the band representing total RPA is shifted due to phosphorylation, loading was normalized against the level of Ku80.
Fig 5.
Analysis of IR-induced DNA damage signaling in cells subjected to pre-IR.
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.
Fig 6.
Promotion of resection by pre-IR treatment is ATM-dependent.
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.