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

The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here we demonstrate that human RIF1 negatively regulates DNA replication by directing Protein Phosphatase 1 (PP1) to limit hosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, finding the regulatory N-terminal domain of MCM4 to be particularly strongly affected. In addition to this role in limiting origin activation, we discover 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 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.