Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair

Although mechanisms of telomere protection are well-defined in differentiated cells,how stem cells sense and respond to telomere dysfunction,in particular telomeric double-strand breaks(DSBs),is poorly characterized.Here,we report the DNA damage signaling,cell cycle,and transcriptome changes in human induced pluripotent stem cells(iPSCs)in response to telomere-internal DSBs.We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres.Using this model,we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response,which leads to p53-independent cell cycle arrest in G2.Using CRISPR–Cas9 to cripple the catalytic domain of telomerase reverse transcriptase,we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres,which instead are effectively repaired by robust homologous recombination(HR).In contrast to HR-based telomere maintenance in mouse embryonic stem cells,where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths,HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length,which is compatible with sustained survival of the cells over several days of TRF1-FokI induction.Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.