Quiescence Induction Triggers Acute Accumulation of DNA Double-Strand Breaks That Drive Quiescence Heterogeneity

DNA double-strand breaks (DSBs) represent one of the most serious threats to genome integrity. Endogenous DSBs chiefly arise from cell cycle activities such as DNA replication and division, while dormant, non-proliferative cells are generally considered protected from such damage. Here we report that induction of quiescence by growth restriction in human retinal pigment epithelial (RPE) cells unexpectedly leads to rapid accumulation of DSBs within hours, reaching levels that exceed those in continuously proliferating cells. These DSBs occur in a cell cycle stage-dependent manner, predominantly in cells past the restriction point when quiescence is induced. Mechanistically, this DSB accumulation results from continued cell cycle activity under growth restriction conditions, accompanied by downregulation of DSB repair genes, allowing these breaks to persist during quiescence. Cells that accumulate more DSBs during quiescence induction enter a deeper state of quiescence, requiring stronger growth stimulation for cell cycle re-entry. Notably, cell cycle re-entry depends on DSB repair mediated by DNA-PK, an intrinsically error-prone process. Our findings establish quiescence induction as a previously unrecognized source of latent genome instability, with implications for tissue maintenance and aging where transitions between quiescence and proliferation are critical.