Orc6 dissociation from chromatin prevents premature loading of MCM at G2 and tetraploid production

DNA replication is tightly regulated to occur only once per cell cycle. The untimely initiation of DNA replication can result in genome instability, leading to aneuploidy, which has been associated with early senescence and cancer. The pre-replication complex, comprising the origin recognition complex (ORC; Orc1-6), Cdc6, Cdt1, and MCM, is required for initiating DNA replication, although the function of Orc6 is yet to be elucidated. Here, we show that Orc6 dissociates from chromatin upon entering the S-phase and that the Orc6 dissociation depends on proteasome activity. Treatment that inhibits proteasome activity, which declines with aging, increases the senescence marker p21 levels, and promotes cell cycle arrest in human immortalized hTERT-RPE1 cells. This treatment induced large nuclei with high levels of chromatin-bound Orc6 and MCM without undergoing mitosis. When the proteasome activity recovered, those cells with high levels of chromatin-bound Orc6 and MCM proceeded to whole-genome DNA replication, confirming that they were tetraploid G1 cells. We propose that proteasome-dependent dissociation of Orc6 from chromatin after S-phase is essential for preventing MCM reloading and the subsequent development of tetraploid cells.