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

Shin-ichiro Hiraga, Tony Ly, Javier Garzon, Zuzana Hořejší, Yoshi-nobu Ohkubo, Akinori Endo, Chikashi Obuse, Simon J. Boulton, Angus I. Lamond, Anne D. Donaldson

Research output: Contribution to journalArticle

51 Citations (Scopus)
7 Downloads (Pure)

Abstract

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.
Original languageEnglish
Article numbere201641983
Pages (from-to)403-419
Number of pages17
JournalEMBO reports
Volume18
Issue number3
Early online date11 Jan 2017
DOIs
Publication statusPublished - 1 Mar 2017

Keywords

  • RIF1
  • PP1
  • origin licensing
  • ORC1
  • MCM

Fingerprint Dive into the research topics of 'Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation'. Together they form a unique fingerprint.

  • Equipment

    Bio-Rad BioPlex 200

    Andrea Holme (Manager)

    Iain Fraser Cytometry Centre

    Research Facilities: Equipment

  • Iain Fraser Cytometry Centre

    Andrea Holme (Manager), Linda Duncan (Senior Application Scientist), Ailsa Laird (Technician) & Kate Burgoyne (Technician)

    Institute of Medical Sciences

    Research Facilities: Facility

  • Activities

    • 1 Invited talk

    Cell cycle flow cytometry identifies human RIF1 protein as a safeguard for DNA replication

    Shinichiro Hiraga (Speaker)

    23 Aug 2017

    Activity: Talk, presentation, public lecture, public engagement, outreach and knowledge exchangeInvited talk

    Cite this