ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.
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.
Adams
Bret R
BR
Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Hawkins
Amy J
AJ
Povirk
Lawrence F
LF
Valerie
Kristoffer
K
D001253:Astrocytes / Q000166:cytology / Q000502:physiology; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D004247:DNA; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000502:physiology*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C508551:NHEJ1 protein, human; D025521:Tumor Suppressor Proteins; C101467:XRCC4 protein, human; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D045643:DNA Repair Enzymes