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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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Description of the NHEJ-red repair cassette and processing of I-SceI-digested DNA.(A) Schematic of the NHEJ-red cassette. (B) Oct3/4 (green) positive hESCs display DsRed (red) 48 h after infection with a multiplicity of infection (MOI) of 30 with Ad-SceI adenovirus. DAPI shows nuclear staining.
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Figure 1: Description of the NHEJ-red repair cassette and processing of I-SceI-digested DNA.(A) Schematic of the NHEJ-red cassette. (B) Oct3/4 (green) positive hESCs display DsRed (red) 48 h after infection with a multiplicity of infection (MOI) of 30 with Ad-SceI adenovirus. DAPI shows nuclear staining.

Mentions: Previous work demonstrated that hESCs are highly proliferative cells with strong G2 checkpoints and an absent G1 checkpoint [26]. For this reason it is believed hESCs would depend extensively on HRR. Indeed, we recently showed that hESCs form RAD51 foci, a marker for HRR, far more extensively and express RAD51 at 10-fold higher levels than differentiated astrocytes [5]. We also demonstrated that the relative fast repair kinetics using γ-H2AX foci as surrogate suggested that hESCs have NHEJ [5]. However, both γ-H2AX and 53BP1 foci resolution was relatively unresponsive to a small molecule inhibitor of DNA-PKcs kinase suggesting that NHEJ in hESCs is largely independent of DNA-PKcs [5]. To determine the nature of NHEJ in hESCs in more detail and to clarify the role of DNA-PKcs, we engineered the hESCs with a lentivirus (LV) carrying an I-SceI repair cassette that would make them more amenable to NHEJ analysis (Figure 1A).


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)

Description of the NHEJ-red repair cassette and processing of I-SceI-digested DNA.(A) Schematic of the NHEJ-red cassette. (B) Oct3/4 (green) positive hESCs display DsRed (red) 48 h after infection with a multiplicity of infection (MOI) of 30 with Ad-SceI adenovirus. DAPI shows nuclear staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984607&req=5

Figure 1: Description of the NHEJ-red repair cassette and processing of I-SceI-digested DNA.(A) Schematic of the NHEJ-red cassette. (B) Oct3/4 (green) positive hESCs display DsRed (red) 48 h after infection with a multiplicity of infection (MOI) of 30 with Ad-SceI adenovirus. DAPI shows nuclear staining.
Mentions: Previous work demonstrated that hESCs are highly proliferative cells with strong G2 checkpoints and an absent G1 checkpoint [26]. For this reason it is believed hESCs would depend extensively on HRR. Indeed, we recently showed that hESCs form RAD51 foci, a marker for HRR, far more extensively and express RAD51 at 10-fold higher levels than differentiated astrocytes [5]. We also demonstrated that the relative fast repair kinetics using γ-H2AX foci as surrogate suggested that hESCs have NHEJ [5]. However, both γ-H2AX and 53BP1 foci resolution was relatively unresponsive to a small molecule inhibitor of DNA-PKcs kinase suggesting that NHEJ in hESCs is largely independent of DNA-PKcs [5]. To determine the nature of NHEJ in hESCs in more detail and to clarify the role of DNA-PKcs, we engineered the hESCs with a lentivirus (LV) carrying an I-SceI repair cassette that would make them more amenable to NHEJ analysis (Figure 1A).

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