Telomeric dysfunction triggers an unstable growth arrest leading to irreparable genomic lesions and entry into cellular senescence

Replicative senescence is the permanent growth arrest caused by gradual telomere attrition occurring at each round of genome replication. Critically shortened telomeres lose their protective shelterin complex and t-loop structure revealing uncapped chromosome ends that are recognized as DNA double-strand breaks causing a p53-dependent DNA damage response (DDR) towards proliferation arrest. Because telomeres are heterogeneous in length within a single cell, the number of short telomeres necessary for senescence onset remains ill defined. Using controlled Tin2-mediated shelterin inactivation, we show that telomere uncapping is not sufficient to trigger senescence. While uncapping generates expected telomeric DNA damage detection, the associated weak DDR allows a rapid bypass of the primary growth arrest and re-entry into the cell cycle despite dysfunctional telomeres. During the ensuing mitosis, fused telomeres lead to additional DNA breaks and to genomic instability including chromosomes bridges or micronuclei, which sustain a secondary entry into stable growth arrest. The loss of p53 prevented both primary and secondary growth arrest, leading to amplified genomic instablility. Our results support a new multistep model for entry into telomere-mediated replicative senescence in normal cells, which is not directly induced by telomere uncapping, but rather by an amplification of DNA lesions caused by telomere fusions that leads to permanent irreparable genome damage.