embr.201641983.pdf (945 kB)
Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation
journal contributionposted on 2020-08-20, 11:19 authored by Shin-ichiro Hiraga, Tony Ly, Javier Garzón, Zuzana Horejsí, Yoshi-nobu Ohkubo, Akinori Endo, Chikashi Obuse, Simon J Boulton, Angus I Lamond, Anne D Donaldson
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 forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation-mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N-terminal domain of MCM4 being 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 the 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.
MCMORC1PP1RIF1origin licensingCell CycleCell Cycle ProteinsChromatinDNA ReplicationHumansMinichromosome Maintenance ProteinsPhosphorylationProteasome Endopeptidase ComplexProtein BindingProtein Interaction Domains and MotifsProtein Phosphatase 1Protein-Serine-Threonine KinasesProteolysisReplication OriginTelomere-Binding ProteinsBoulton FC0010480601 Biochemistry and Cell BiologyDevelopmental Biology