title: Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation.
abstract: 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.
authors.affiliation: Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, UK s.hiraga@abdn.ac.uk a.d.donaldson@abdn.ac.uk.
authors.forename: Shin-Ichiro
authors.identifier: 0000-0002-8722-3869
authors.initials: SI
authors.lastname: Hiraga
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Ly
authors.affiliation: Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, UK.
authors.forename: Javier
authors.initials: J
authors.lastname: Garzón
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratories, South Mimms, UK.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Hořejší
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Yoshi-Nobu
authors.initials: YN
authors.lastname: Ohkubo
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Akinori
authors.initials: A
authors.lastname: Endo
authors.affiliation: Graduate School of Life Science, Hokkaido University, Sapporo Hokkaido, Japan.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Obuse
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratories, South Mimms, UK.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Boulton
authors.affiliation: Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Angus I
authors.identifier: 0000-0001-6204-6045
authors.initials: AI
authors.lastname: Lamond
authors.affiliation: Institute of Medical Sciences, School of Medicine, Medical Sciences & Nutrition, University of Aberdeen, Aberdeen, UK s.hiraga@abdn.ac.uk a.d.donaldson@abdn.ac.uk.
authors.forename: Anne D
authors.identifier: 0000-0001-7842-8136
authors.initials: AD
authors.lastname: Donaldson
mesh_terms: D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D054645:Protein Phosphatase 1 / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D059748:Proteolysis; D018741:Replication Origin; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; C492743:Rif1 protein, human; D034501:Telomere-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D054645:Protein Phosphatase 1; D046988:Proteasome Endopeptidase Complex; D064110:Minichromosome Maintenance Proteins
keywords: 
  MCM
  ; ORC1; PP1; RIF1; origin licensing