The DNA damage response pathway is required for multiciliated cell differentiation.

DNA damage can result from external sources or occur during programmed genome rearrangements in processes like immunity or meiosis. To maintain genome integrity, cells activate DNA repair pathways that prevent harmful outcomes such as cancer or immune dysfunction. In this study, we uncover a novel role for DNA damage during the terminal differentiation of multiciliated cells (MCCs). MCCs, which line the airways, reproductive tracts, and brain ventricles, produce hundreds of motile cilia, each anchored by a centriole. Therefore, MCCs must generate hundreds of centrioles during differentiation. Normally, centriole duplication is tightly linked to the S and G2 phases of the cell cycle, raising questions about how MCCs override numerical and temporal restrictions on centriole duplication. We find that differentiating MCCs accumulate extensive double-stranded DNA breaks during centriole amplification, with damage levels correlating with centriole number. DNA damage response (DDR) kinases are essential for supporting centriole biogenesis and ciliogenesis. We also observe that transcriptional activity, required for the expression of centriole and cilia genes, produces RNA-DNA hybrids (R-loops) that co-localize with DNA damage. This suggests that transcription-coupled DNA damage helps initiate a pseudo-cell cycle program, permitting centriole amplification without triggering full S/G2 phase processes. Our findings indicate that MCCs harness DDR signaling as part of their developmental program, revealing a broader principle in which the canonical cell cycle is adaptively rewired during differentiation.