Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

The RIF1 protein controls DNA replication, but it has been unclear whether the molecular mechanisms are conserved throughout eukaryotes. Yeast RIF1 negatively regulates DNA replication by directing Protein Phosphatase 1 (PP1) to limit phosphorylation-mediated activation of the MCM replicative helicase. Here we demonstrate that human RIF1 interacts with all three isoforms of human PP1 to prevent MCM complex hyperphosphorylation. RIF1 limits phosphorylation of the MCM2, MCM3, MCM4, and MCM6 helicase subunits, with particularly marked effect on the regulatory N-terminal domain of MCM4. In addition to this role in limiting origin activation, we identify an unexpected new role for human RIF1-PP1 in mediating efficient origin licensing. Specifically, during G1 phase of the cell cycle RIF1-PP1 protects the origin binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1-depleted cells exhibit somewhat increased spacing between active origins. Human RIF1 therefore acts as a PP1-targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.