Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

染色体分离依赖于着丝粒，但它们的重复 DNA 在病理条件下往往容易发生异常重排。维持着丝粒完整性以防止着丝粒相关染色体易位的因素尚不清楚。在这里，我们证明了着丝粒特异性组蛋白 H3 变体 CENP-A 在保护 α 卫星重复序列的 DNA 复制以防止结构非整倍性方面的重要性。在 S 期快速去除 CENP-A，而不是其他细胞周期阶段，导致 R 环积累，着丝粒转录物增加，并干扰复制叉进程。没有 CENP-A 的复制会导致 alpha 卫星以 R 环依赖的方式重组、未完成的复制和后期桥接。反过来，染色体断裂和易位特别出现在着丝粒区域。我们的研究结果提供了关于特殊着丝粒染色质如何在 S 期维持转录的非编码重复 DNA 完整性的见解。