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. 2010 Apr 2;5(4):e10001.
doi: 10.1371/journal.pone.0010001.

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants

Bret R Adams  1 , Sarah E Golding, Raj R Rao, Kristoffer Valerie
Affiliations

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants

Bret R Adams et al. PLoS One. .

Abstract

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of repair foci formation and resolution in hESCs, neural progenitors and astrocytes.
A. Depicted is immunostaining of hESCs after treatment with 0, 0.5, and 2 Gy and exposed to p-(S1981) ATM antibody 15 min after radiation. B. Immunostaining of 53BP1, p-(S1981) ATM, and γ-H2AX foci 15 min after irradiation in hESCs after doses from 0.1 to 5 Gy. n = 30 cells per time point. C. Graphical depiction of the number of γ-H2AX foci in hESCs, NPs and astrocytes exposed to 2 Gy over 24 h. Data are expressed as mean IRIF per cell. n = 100 cells per time point. There was <1 focus on average in the untreated hESC, NP and astrocyte populations (shown as symbols at time 0). Error bars indicate the standard error of the mean (SEM). Asterisks indicate statistical significance between the number of foci observed in hESCs, NPs, and astrocytes at the same time point.
Figure 2
Figure 2. Differentiation affects RAD51 foci formation and expression.
A. Graphical depiction of the percentage of RAD51 foci in hESCs, NPs, and astrocytes after 2 Gy. Cells were scored either as positive (>1 focus) or negative (≤1 foci) for RAD51. Errors bars, SEM for three independent data sets with n>200. There were statistically significant (p<0.05) increases in the number of cells with RAD51 foci after radiation compared to unirradiated cells at all time points for hESCs and NPs whereas there was no statistical change in the astrocytes. B. Western blot showing expression of RAD51 in hESC, NPs and astrocytes. Fold depicts relative differences in RAD51 levels of the 37-kDa form after normalization to β-actin which served as a loading control.
Figure 3
Figure 3. ATM kinase is important for γ-H2AX foci resolution in astrocytes but not in hESCs.
Graphical depiction (left panel) and images (right panel) of γ-H2AX foci in hESCs, NPs, and astrocytes exposed to 10 µM KU-55933 A. 1 h prior to or B. 15 min after radiation with 2 Gy. There was <1 focus on average in the untreated hESC, NP and astrocyte populations (shown as symbols at time 0). Asterisks indicate statistical significance between the data sets generated with NPs and astrocytes, respectively, with KU-55933 present or not at the same time point. Data points, foci per nucleus; Error bars, SEM for data sets n = 50.
Figure 4
Figure 4. ATM kinase is functioning in hESCs.
Western blot of extracts isolated from hESCs 15 min after exposure to 5 Gy with or without KU-55933 (10 µM) added 1 h prior to radiation. Fold change depicts phosphorylation of CHK2 (T68) and H2AX (S139) after normalization to β-actin which served as a loading control.
Figure 5
Figure 5. ATR is required for DSB repair in hESCs.
A. Graphical depiction of γ-H2AX (top panel), and RAD51 (bottom panel) foci determined in the presence of 0, 2, 4, and 8 mM caffeine administered 1 h prior to radiation (2 Gy). Asterisks designate statistical significance between the two data sets adjacent to the asterisk at the same time point. Data points, foci per nucleus; Error bars, SEM for data sets n = 50. B. Graphical depiction of ATR mRNA levels normalized to GAPDH mRNA after transfection with GFP control siRNAs and siRNAs targeting ATR. Experiments were performed in triplicate, and the SEM indicated by error bars. C. Western blot showing ATR expression 48 h after transfection of BGO1V cells with GFP control siRNAs or siRNAs targeting ATR. The fold change in ATR was calculated after normalization to DNA-PKcs which served as a loading control. D. Graphical depiction of the effects of ATR knockdown on DSB repair determined by γ-H2AX foci. Cells were transfected with GFP control siRNAs or siRNAs targeting ATR, irradiated with 2 Gy 48 h after transfection, and fixed 6 h later. Data points, γ-H2AX foci per nucleus; Error bars, SEM for data sets n = 100. E. Representative images of cells with γ-H2AX foci (red) with DAPI staining showing nuclei (blue).
Figure 6
Figure 6. Astrocytes rely completely on NHEJ.
A. Graphical depiction of γ-H2AX (top panel) and 53BP1 (bottom panel) foci in astrocytes after radiation. Cells were treated or not with KU-55933 (10 µM), KU-57788 (2.5 µM) or both for 1 h followed by irradiation with 2 Gy. B. Graphical depiction of γ-H2AX (top panel) and 53BP1 (bottom panel) foci in BGO1V cells treated or not with KU-57788 (2.5 µM) followed by radiation with 2 Gy. Asterisks designate statistical significance between data sets of untreated and drug-treated cells at the same time point. There was <1 focus on average in the untreated hESC, NP and astrocyte populations. Data points, foci per nucleus; Error bars, SEM for data sets n = 50.
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