Proteasome-dependent Orc6 removal from chromatin upon S-phase entry safeguards against MCM reloading and tetraploidy

DNA replication is tightly regulated to occur only once per cell cycle, as untimely re-initiation can lead to aneuploidy, which is associated with early senescence and cancer. The pre-replication complex (comprising Orc1-6, Cdc6, Cdt1, and MCM) is essential for the initiation of DNA replication, but the dynamics and function of Orc6 during the cell cycle remain elusive. Here, we demonstrate that Orc6 associates with chromatin during G1-phase and dissociates upon S-phase entry. The dissociation of Orc6 from chromatin is dependent on proteasome activity, and inhibition of the proteasome leads to the accumulation of chromatin-bound Orc6, which promotes abnormal MCM loading after S-phase entry without undergoing mitosis in human immortalized hTERT-RPE1 cells. Following release from proteasome inhibition, cells with elevated levels of chromatin-bound Orc6 and MCM proceed to the next replication phase as tetraploid cells. Our findings suggest that the proteasome-dependent dissociation of Orc6 after DNA replication is critical for preventing inappropriate MCM reloading and tetraploid formation.