Compromised DNA replication in gut cells underlies sensitivity to genotoxic stress in the tardigrade Hypsibius exemplaris

Tardigrades withstand severe DNA insults, including extreme doses of ionising radiation, through unique protective proteins and strong upregulation of canonical DNA repair pathways. Yet these extremophile animals are not immortal, and the cellular and organismal processes that ultimately fail under sustained genotoxic stress have not been characterised. Here, we identify DNA replication as the key vulnerability in the tardigrade Hypsibius exemplaris. Using the radiomimetic drugs bleomycin or zeocin to induce DNA breaks, we show that continuous exposure progressively kills tardigrades, accompanied by striking body shrinkage and lipid depletion. DNA synthesis labelling reveals that these drugs disrupt replication and trigger de novo reparative synthesis in select non-dividing tissues. Pulse-wash experiments demonstrate that even transient genotoxic exposure coincident with cell replication irreversibly exhausts gut cell proliferative capacity, leading to midgut failure and animal death, despite systemic induction of DNA repair genes. Germ cells and embryos, with their high proliferation rates, show heightened sensitivity. Cross-phyla survival assays in the eutelic nematode Caenorhabditis elegans and neoblast-rich planarian Schmidtea mediterranea further link proliferative activity to mortality kinetics under DNA damage. Collectively, our findings pinpoint DNA replication as an Achilles' heel of organismal survival under genotoxic stress, even in animals renowned for their extraordinary DNA damage tolerance.