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 DNA 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 DDR, 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.