Limits...
Pre-exposure to ionizing radiation stimulates DNA double strand break end resection, promoting the use of homologous recombination repair.

Nakajima NI, Hagiwara Y, Oike T, Okayasu R, Murakami T, Nakano T, Shibata A - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Charged Particle Therapy and International Open Laboratory, National Institute of Radiological Sciences, Chiba, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4380452&req=5

pone.0122582.g001: 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.

Mentions: To investigate whether pre-IR treatment affects the balance of DSB repair-pathway choice between HR and NHEJ, we measured the efficiency of repair following pre-IR using chromosomal HR and NHEJ assays in the U2OS and H1299 cell lines, respectively (Fig 1A) [25,26]. To confirm that each assay monitored HR or NHEJ as predicted, we examined repair efficiencies in cells depleted for CtIP or BRCA2 using specific siRNAs or treated with a DNA-PKcs inhibitor. CtIP is essential for initiation of DSB end resection in HR [27], and BRCA2 plays an important role in HR by delivering and loading RAD51 onto single-stranded DNA. On the other hand, the kinase activity of DNA-PKcs is required for NHEJ. As expected, HR efficiency was significantly reduced in CtIP- or BRCA2-depleted cells, and NHEJ efficiency was decreased by the DNA-PKcs inhibitor, demonstrating that each assay is monitoring the desired pathway (HR or NHEJ), as expected (S1 Fig). To analyze the impact of pre-IR treatment on the choice of chromosomal DSB repair pathway, we treated cells with 0.1 or 0.2 Gy IR 2 or 6 h before I-SceI induction (Fig 1B). Because irradiation doses greater than 0.5 Gy irradiation may lead to checkpoint arrest or cell death, in this study we treated cells with doses of 0.2 Gy or less (at these doses, no DSBs persist >6 h post-IR) [28]. Cells irradiated with 0.1 or 0.2 Gy IR 6 h prior to I-SceI transfection exhibited elevated rates of HR, whereas pre-IR at 2 h or 12–24 h did not significantly affect HR (Fig 1C and 1D). Together, these results indicate that the rate of HR usage is elevated ~8 h after pre-IR treatment. The timing of pre-IR seemed to be important for this promotion of HR. On the other hand, the rate of NHEJ was slightly reduced by pre-IR, although this effect was not statistically significant (Fig 1C) (N.B.: because the efficiencies of HR and NHEJ were measured in separate assay systems, the rates of each type of repair are not linked).


Pre-exposure to ionizing radiation stimulates DNA double strand break end resection, promoting the use of homologous recombination repair.

Nakajima NI, Hagiwara Y, Oike T, Okayasu R, Murakami T, Nakano T, Shibata A - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380452&req=5

pone.0122582.g001: 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.
Mentions: To investigate whether pre-IR treatment affects the balance of DSB repair-pathway choice between HR and NHEJ, we measured the efficiency of repair following pre-IR using chromosomal HR and NHEJ assays in the U2OS and H1299 cell lines, respectively (Fig 1A) [25,26]. To confirm that each assay monitored HR or NHEJ as predicted, we examined repair efficiencies in cells depleted for CtIP or BRCA2 using specific siRNAs or treated with a DNA-PKcs inhibitor. CtIP is essential for initiation of DSB end resection in HR [27], and BRCA2 plays an important role in HR by delivering and loading RAD51 onto single-stranded DNA. On the other hand, the kinase activity of DNA-PKcs is required for NHEJ. As expected, HR efficiency was significantly reduced in CtIP- or BRCA2-depleted cells, and NHEJ efficiency was decreased by the DNA-PKcs inhibitor, demonstrating that each assay is monitoring the desired pathway (HR or NHEJ), as expected (S1 Fig). To analyze the impact of pre-IR treatment on the choice of chromosomal DSB repair pathway, we treated cells with 0.1 or 0.2 Gy IR 2 or 6 h before I-SceI induction (Fig 1B). Because irradiation doses greater than 0.5 Gy irradiation may lead to checkpoint arrest or cell death, in this study we treated cells with doses of 0.2 Gy or less (at these doses, no DSBs persist >6 h post-IR) [28]. Cells irradiated with 0.1 or 0.2 Gy IR 6 h prior to I-SceI transfection exhibited elevated rates of HR, whereas pre-IR at 2 h or 12–24 h did not significantly affect HR (Fig 1C and 1D). Together, these results indicate that the rate of HR usage is elevated ~8 h after pre-IR treatment. The timing of pre-IR seemed to be important for this promotion of HR. On the other hand, the rate of NHEJ was slightly reduced by pre-IR, although this effect was not statistically significant (Fig 1C) (N.B.: because the efficiencies of HR and NHEJ were measured in separate assay systems, the rates of each type of repair are not linked).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Charged Particle Therapy and International Open Laboratory, National Institute of Radiological Sciences, Chiba, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus