Dephosphorylation of the pre-initiation complex during S-phase is critical for origin firing

Genome stability requires complete DNA duplication exactly once before cell division. In eukaryotes, cyclin-dependent kinase (CDK) plays a dual role in this regulation by inhibiting helicase loading factors before also activating origin firing. CDK activates initiation by phosphorylation of two substrates, Sld2 and Sld3, forming a transient and limiting intermediate - the pre-initiation complex (pre-IC). The importance and mechanism of dissociation of the pre-IC from origins is not understood. Here we show in the budding yeast Saccharomyces cerevisiae that CDK phosphorylation of Sld3 and Sld2 is specifically and rapidly turned over during interphase by the PP2A and PP4 phosphatases. Inhibiting dephosphorylation of Sld3/Sld2 causes dramatic defects in replication initiation genome-wide, retention of the pre-IC at origins and cell death. These studies not only provide a mechanism to guarantee that Sld3 and Sld2 are dephosphorylated before helicase loading factors but also uncover a novel positive role for phosphatases in eukaryotic origin firing.