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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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High-fidelity NHEJ decreases through differentiationBG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h after infection. DNA was amplified with Amplitaq Gold and was digested with PsiI endonuclease where indicated. (Columns) High-fidelity NHEJ levels were determined by the relative level of the digested portion (PsiI-sensitive) of the PCR DNA fragment as a fraction of uncut DNA; (Error bars) SEM for three samples. Fold (x) indicates changes in relative repair levels when compared to the astrocyte samples.
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Figure 7: High-fidelity NHEJ decreases through differentiationBG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h after infection. DNA was amplified with Amplitaq Gold and was digested with PsiI endonuclease where indicated. (Columns) High-fidelity NHEJ levels were determined by the relative level of the digested portion (PsiI-sensitive) of the PCR DNA fragment as a fraction of uncut DNA; (Error bars) SEM for three samples. Fold (x) indicates changes in relative repair levels when compared to the astrocyte samples.

Mentions: If hESCs rely on alternative forms of repair it is possible there would be a difference in the fidelity with which the repair occurs. As hESCs differentiated to NPs and astrocytes there was a progressive decrease in the extent to which the overhangs were filled in with AA nucleotides indicating high-fidelity NHEJ. hESCs displayed a 2.6-fold higher levels of high-fidelity NHEJ compared to astrocytes, whereas NPs displayed a 1.8-fold increase compared to astrocytes (Figure 7 and Figure S1). A U87 glioma cell clone carrying the NHEJ-red vector [8], showed a 1.3-fold higher level of high-fidelity NHEJ (data not shown) compared to in vitro derived astrocytes. In order to verify the results that the ability of PsiI to digest the 125-bp PCR product corresponds to high-fidelity NHEJ, DNA sequencing of the cloned PCR fragments was performed. DNA sequencing revealed that 50% of the amplified DNA showed the presence of the PsiI site (Table 1 and Table S1), which correlates well with the ~55% obtained by PsiI digestion (Figure 7). DNA changes included small deletions of 1-3 nucleotides but no larger deletions or any insertions were noted. In line with this finding, cloning and sequencing of PCR fragments from the U87/NHEJ-red cells showed a similar correlation between PsiI digestion and DNA sequence analysis (data not shown). Altogether, high-fidelity repair correlates with replicative growth and cell cycle distribution and was close to 3-fold higher in hESCs than in astrocytes and human glioma cells. Furthermore, our data suggest that when presented as an option partially complementary DNA overhangs are repaired without resection.


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)

High-fidelity NHEJ decreases through differentiationBG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h after infection. DNA was amplified with Amplitaq Gold and was digested with PsiI endonuclease where indicated. (Columns) High-fidelity NHEJ levels were determined by the relative level of the digested portion (PsiI-sensitive) of the PCR DNA fragment as a fraction of uncut DNA; (Error bars) SEM for three samples. Fold (x) indicates changes in relative repair levels when compared to the astrocyte samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: High-fidelity NHEJ decreases through differentiationBG01V/-, NP/-, and astrocyte/NHEJ-red cells were infected with Ad-SceI and collected 24 h after infection. DNA was amplified with Amplitaq Gold and was digested with PsiI endonuclease where indicated. (Columns) High-fidelity NHEJ levels were determined by the relative level of the digested portion (PsiI-sensitive) of the PCR DNA fragment as a fraction of uncut DNA; (Error bars) SEM for three samples. Fold (x) indicates changes in relative repair levels when compared to the astrocyte samples.
Mentions: If hESCs rely on alternative forms of repair it is possible there would be a difference in the fidelity with which the repair occurs. As hESCs differentiated to NPs and astrocytes there was a progressive decrease in the extent to which the overhangs were filled in with AA nucleotides indicating high-fidelity NHEJ. hESCs displayed a 2.6-fold higher levels of high-fidelity NHEJ compared to astrocytes, whereas NPs displayed a 1.8-fold increase compared to astrocytes (Figure 7 and Figure S1). A U87 glioma cell clone carrying the NHEJ-red vector [8], showed a 1.3-fold higher level of high-fidelity NHEJ (data not shown) compared to in vitro derived astrocytes. In order to verify the results that the ability of PsiI to digest the 125-bp PCR product corresponds to high-fidelity NHEJ, DNA sequencing of the cloned PCR fragments was performed. DNA sequencing revealed that 50% of the amplified DNA showed the presence of the PsiI site (Table 1 and Table S1), which correlates well with the ~55% obtained by PsiI digestion (Figure 7). DNA changes included small deletions of 1-3 nucleotides but no larger deletions or any insertions were noted. In line with this finding, cloning and sequencing of PCR fragments from the U87/NHEJ-red cells showed a similar correlation between PsiI digestion and DNA sequence analysis (data not shown). Altogether, high-fidelity repair correlates with replicative growth and cell cycle distribution and was close to 3-fold higher in hESCs than in astrocytes and human glioma cells. Furthermore, our data suggest that when presented as an option partially complementary DNA overhangs are repaired without resection.

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