Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of Geminin through its N-terminal region

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.