Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis

Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell-cycle-dependent changes in its Orc1 subunit. Here, we show that Orc1 modifications such as monoubiquitylation and hyperphosphorylation that occur normally during S and G2-M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of pre-replication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induced p53-independent apoptosis, and Orc1 accumulated perinuclearly rather than uniformly throughout the nucleus. This behavior mimicked the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that did not induce apoptosis, prevented induction of apoptosis and restored uniform nuclear localization of Orc1. This would promote assembly of ORC-chromatin sites, such as occurs during the transition from M to G1 phase. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of 'unmodified' Orc1.