Oxidative DNA damage drives centromere fragility and chromosome instability.

Centromeres are essential regions of the genome that ensure chromosome segregation during mitosis but are also hotspots for chromosome breaks and rearrangements in cancer. The mechanisms underlying this fragility are not fully elucidated. Here, we show that oxidative DNA damage destabilizes centromeres and promotes chromosome instability. Using a chemoptogenetic system to generate singlet oxygen locally at centromeres, we uncouple centromeric oxidative damage from global oxidative stress. Oxidative base lesions slow DNA synthesis, destabilize CENP-A chromatin, and generate DNA breaks that persist into subsequent cell cycles. Single-cell time-lapse imaging reveals cell-cycle-dependent outcomes, with lesions before or during replication favoring quiescence and post-replicative lesions allowing mitotic progression but compromising daughter-cell proliferation. Finally, in pre-tumorigenic cells, centromeric oxidative lesions lead to mitotic defects, aneuploidy, and whole-arm chromosome translocations. Collectively, we identify centromeres as cell-cycle-sensitive DNA damage sensors and oxidative stress as a direct driver of centromere fragility and chromosome instability.