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

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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

Fingerprint

Protein Phosphatase 1
Replication Origin
Licensure
DNA Replication
Chemical activation
Phosphorylation
DNA
Multicarrier modulation
Origin Recognition Complex
Cell Cycle Proteins
G1 Phase
Proteasome Endopeptidase Complex
Eukaryota
Yeast
Carrier Proteins
Protein Isoforms
Yeasts
Degradation
Proteins

Keywords

  • RIF1
  • PP1
  • origin licensing
  • ORC1
  • MCM

Cite this

Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation. / Hiraga, Shin-ichiro; Ly, Tony; Garzon, Javier; Hořejší, Zuzana; Ohkubo, Yoshi-nobu; Endo, Akinori; Obuse, Chikashi; Boulton, Simon J.; Lamond, Angus I.; Donaldson, Anne D.

In: EMBO reports, Vol. 18, No. 3, e201641983, 01.03.2017, p. 403-419.

Research output: Contribution to journalArticle

Hiraga, Shin-ichiro ; Ly, Tony ; Garzon, Javier ; Hořejší, Zuzana ; Ohkubo, Yoshi-nobu ; Endo, Akinori ; Obuse, Chikashi ; Boulton, Simon J. ; Lamond, Angus I. ; Donaldson, Anne D. / Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation. In: EMBO reports. 2017 ; Vol. 18, No. 3. pp. 403-419.
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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.",
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AU - Obuse, Chikashi

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AU - Lamond, Angus I.

AU - Donaldson, Anne D.

N1 - We thank David Stead at the Aberdeen Proteomics Service for help in mass spectrometry interpretation, and Raif Yücel and his team at the University of Aberdeen Iain Fraser Cytometry Centre for assistance with flow cytometry. We thank Robert Alver and Julian Blow at University of Dundee for advice on the use of tautomycetin. Peter Cherepanov of the Francis Crick Institute gifted XL413. Daniel Durocher of Lunenfeld-Tanenbaum Research Institute gifted DNA constructs. Work by ADD and SH was supported by Cancer Research UK Grant A13356, Cancer Research UK Programme Award A19059, and BBSRC grant (BB/K006304/1). AIL was supported by Wellcome Trust Awards (108058/Z/15/Z & 105024/Z/14/Z). This work was also supported by JSPS KAKENHI Grant # 16H04739, 25116004 to CO and 16J04327 to YO.

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N2 - 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.

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