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.
Highlights
Human embryonic stem cells are notable because they possess the ability to self-renew indefinitely and are capable of differentiating into all tissues of an organism
We report here that rapidly proliferating Human embryonic stem cells (hESCs) utilize nonhomologous end joining (NHEJ) in a process that is Ataxia telangiectasia mutated (ATM)-independent and largely DNA-PKcs-independent and show that upon differentiation of the hESCs to neural progenitors (NPs) and astrocytes, the rate of NHEJ progressively increases whereas the fidelity of repair decreases
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]
Summary
Human embryonic stem cells (hESCs) are notable because they possess the ability to self-renew indefinitely and are capable of differentiating into all tissues of an organism. These cells are able to preserve their genomic and epigenetic integrity to a higher degree than somatic cells [1]. ESCs may use several mechanisms to maintain genomic stability including the up-regulation of DNA repair, the utilization of highfidelity forms of repair, and the efficient elimination of damaged cells by apoptosis [2,3,4,5]. The form of DSB repair with the highest fidelity is HRR which utilizes homologous sequences from a sister chromatid, homologous chromosome, or repetitive sequence as templates for repairing the damaged DNA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
