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ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

Adams BR, Hawkins AJ, Povirk LF, Valerie K - Aging (Albany NY) (2010)

Bottom Line: ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes.The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown.Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

ABSTRACT
We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

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NHEJ occurs with faster kinetics after terminal differentiation.(A) hESCs, NPs and astrocytes were seeded and 12 h later infected with Ad-SceI at an MOI of 100. Expression of HA-tagged I-SceI was examined in samples harvested 24 h after infection. (B) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h later. (Columns) Relative NHEJ levels were determined by genomic DNA qPCR and normalized to β-actin levels; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. *p < 0.05: **p < 0.01; ***p < 0.001. (C) (Top Panel) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-I-SceI at an MOI of 30. DsRed events were determined by FACS 48 and 72 h after infection. Fold (x) and statistical significance indicates changes in the relative repair levels when compared to the hESC sample. (Columns) % DsRed+ cells with 60,000 events collected; (Error bars) SEM for three independent experiments. (Bottom Panel) Representative FACS images of DsRed+ cells at 72 h after infection.
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Figure 3: NHEJ occurs with faster kinetics after terminal differentiation.(A) hESCs, NPs and astrocytes were seeded and 12 h later infected with Ad-SceI at an MOI of 100. Expression of HA-tagged I-SceI was examined in samples harvested 24 h after infection. (B) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h later. (Columns) Relative NHEJ levels were determined by genomic DNA qPCR and normalized to β-actin levels; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. *p < 0.05: **p < 0.01; ***p < 0.001. (C) (Top Panel) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-I-SceI at an MOI of 30. DsRed events were determined by FACS 48 and 72 h after infection. Fold (x) and statistical significance indicates changes in the relative repair levels when compared to the hESC sample. (Columns) % DsRed+ cells with 60,000 events collected; (Error bars) SEM for three independent experiments. (Bottom Panel) Representative FACS images of DsRed+ cells at 72 h after infection.

Mentions: Previous work from our group established optimal conditions for the growth and differentiation of hESCs on feeder-free cultures into NPs and astrocytes [5,27,28]. We have not only shown a loss in proliferation after differentiation to astrocytes, but also changes in morphological and phenotypic properties such as increased glutamate uptake associated with astrocytes [27]. Since these cell populations are identical at the genetic level any changes observed are likely due to alterations in epigenetics. Thus, it is possible that adenovirus infection and I-SceI expression may change through differentiation thus accounting for the differences seen in NHEJ. To determine the relative levels of I-SceI expression in these cell populations, hESCs, NPs, and astrocytes were infected with an equal MOI of adenovirus expressing HA-tagged I-SceI. These three cell populations expressed very similar levels of HA-SceI (Figure 3A). This assay would therefore be able to accurately assess any changes in NHEJ repair through differentiation.


ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

Adams BR, Hawkins AJ, Povirk LF, Valerie K - Aging (Albany NY) (2010)

NHEJ occurs with faster kinetics after terminal differentiation.(A) hESCs, NPs and astrocytes were seeded and 12 h later infected with Ad-SceI at an MOI of 100. Expression of HA-tagged I-SceI was examined in samples harvested 24 h after infection. (B) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h later. (Columns) Relative NHEJ levels were determined by genomic DNA qPCR and normalized to β-actin levels; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. *p < 0.05: **p < 0.01; ***p < 0.001. (C) (Top Panel) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-I-SceI at an MOI of 30. DsRed events were determined by FACS 48 and 72 h after infection. Fold (x) and statistical significance indicates changes in the relative repair levels when compared to the hESC sample. (Columns) % DsRed+ cells with 60,000 events collected; (Error bars) SEM for three independent experiments. (Bottom Panel) Representative FACS images of DsRed+ cells at 72 h after infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: NHEJ occurs with faster kinetics after terminal differentiation.(A) hESCs, NPs and astrocytes were seeded and 12 h later infected with Ad-SceI at an MOI of 100. Expression of HA-tagged I-SceI was examined in samples harvested 24 h after infection. (B) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h later. (Columns) Relative NHEJ levels were determined by genomic DNA qPCR and normalized to β-actin levels; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. *p < 0.05: **p < 0.01; ***p < 0.001. (C) (Top Panel) BG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-I-SceI at an MOI of 30. DsRed events were determined by FACS 48 and 72 h after infection. Fold (x) and statistical significance indicates changes in the relative repair levels when compared to the hESC sample. (Columns) % DsRed+ cells with 60,000 events collected; (Error bars) SEM for three independent experiments. (Bottom Panel) Representative FACS images of DsRed+ cells at 72 h after infection.
Mentions: Previous work from our group established optimal conditions for the growth and differentiation of hESCs on feeder-free cultures into NPs and astrocytes [5,27,28]. We have not only shown a loss in proliferation after differentiation to astrocytes, but also changes in morphological and phenotypic properties such as increased glutamate uptake associated with astrocytes [27]. Since these cell populations are identical at the genetic level any changes observed are likely due to alterations in epigenetics. Thus, it is possible that adenovirus infection and I-SceI expression may change through differentiation thus accounting for the differences seen in NHEJ. To determine the relative levels of I-SceI expression in these cell populations, hESCs, NPs, and astrocytes were infected with an equal MOI of adenovirus expressing HA-tagged I-SceI. These three cell populations expressed very similar levels of HA-SceI (Figure 3A). This assay would therefore be able to accurately assess any changes in NHEJ repair through differentiation.

Bottom Line: ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes.The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown.Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

ABSTRACT
We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

Show MeSH
Related in: MedlinePlus