Centromeric α-satellite DNA is a hotspot of genotoxic damage, incomplete repair, and cytoplasmic mislocalization.

Centromeric α-satellite DNA constitutes a highly repetitive and structurally specialized component of the human genome, yet the mechanisms underlying its damage susceptibility and repair fidelity under genotoxic stress remain undefined. Here, we demonstrate that genotoxic stress preferentially targets active centromeres, generating DNA double-strand breaks (DSBs) within α-satellite arrays. Using bleomycin as a defined genotoxic perturbation, we identify dynamic alterations in centromeric repeat content, manifesting as net copy number losses and gains across multiple chromosome-specific α-satellite arrays following damage. Similar centromere-associated damage signatures are observed in fibroblasts from patients with limited cutaneous systemic sclerosis, indicating that these features extend beyond experimental systems. Centromeric DSBs engage ATM-dependent DNA damage signaling and are repaired predominantly through RAD51-associated homologous recombination; however, repair fails to fully restore centromeric integrity. This incomplete repair is associated with defects in kinetochore organization, chromosome missegregation, and the formation of micronuclei containing centromeric DNA. Notably, ~30% of these structures retain CENP-B but lacks detectable CENP-A, indicating disruption of centromere chromatin organization. Centromeric chromatin is frequently mislocalized to the cytoplasm following nuclear envelope perturbation, where immunofluorescence analysis reveals proximity to MHC class II (HLA-DRB1). Together, these findings establish centromeric α-satellite DNA as a vulnerability hotspot under genotoxic stress, with implications for chromosome instability and chromatin antigen exposure in fibrosis-associated autoimmunity.