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

Genome stability requires complete DNA duplication exactly once before division. In eukaryotes, cyclin-dependent kinase (CDK) plays a dual role in this regulation by inhibiting helicase loading factors and activating origin firing. CDK drives initiation by phosphorylation of two substrates, Sld2 and Sld3, forming the transient and limiting intermediate called 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 phosphorylations of Sld3 and Sld2 are specifically and rapidly turned-over during interphase by the PP2A and PP4 phosphatases. Inhibition of 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 ensure that Sld3/Sld2 are dephosphorylated before helicase loading factors but also establish a novel positive role for phosphatases in eukaryotic origin firing. Overall design: Examination of replication progession and CHIP of Mcm4-3HA and Sld2-6HA by high-throughput sequencing of DNA in two strains (swe1? osTir1 cdc55-AID sld3-13myc; swe1? osTir1 cdc55-AID pph3-AID sld3-R(2A)-13myc) from G1 to S phase.