Abstract
DNA replication origin licensing, the process of MCM helicase loading, is an early essential step in replication and should be restricted to G1 phase to avoid re-replication and genome instability. Cdt1 is a critical MCM loading factor whose licensing activity must be restrained after G1. We discovered that Cdt1 hyperphosphorylation during G2 and M phase is essential to prevent re-replication and DNA damage, the first example of direct Cdt1 licensing activity control by post-translational modification. This hyperphosphorylation specifically requires Cyclin A/CDK1 and occurs at a cluster of phosphorylation sites in a disordered Cdt1 linker region. Hyperphosphorylation interferes with Cdt1-MCM binding independently of protein degradation or inhibition by the Cdt1 inhibitor, Geminin. At the M-G1 transition, Cdt1 is re-activated by protein phosphatase 1-dependent dephosphorylation. We propose that distinct, non-redundant re-replication inhibition mechanisms act in a sequential relay from early S through mitosis to ensure once, and only once, chromosome duplication.