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,it is poorly understood how stem cells sense and respond to telomere dysfunction.In particular,the broader impact of telomeric double-strand breaks(DSBs)in these cells is poorly characterized.Here,we report on deoxyribonucleic acid damage signaling,cell cycle,and transcriptome-level changes in human induced pluripotent stem cells(iPSCs)in response to telomere-internal DSBs.We engineered 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,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,we find neither evidence that HR causes extension of telomeres beyond their initial lengths,nor an apparent role for ZSCAN4 in this process.Rather,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 damage.