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

Shin‐Ichiro Hiraga,Chikashi Obuse,Simon J Boulton,Yoshi‐Nobu Ohkubo,Zuzana Hořejší,Anne D Donaldson,Akinori Endo,Javier Garzón,Tony Ly,Angus I Lamond

doi:10.15252/embr.201641983

Shin‐Ichiro Hiraga, Chikashi Obuse + Show 8 more

Open Access

https://doi.org/10.15252/embr.201641983

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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.

Highlights

The process of DNA replication is critical during cell proliferation for daughter cells to inherit a complete set of genomic information.DNA replication is tightly controlled to prevent either under- or over-replication, with the establishment of licensed DNA replication origins and their subsequent activation closely coupled with other cell cycle events.Best understood in budding yeast, the mechanisms controlling origin establishment and activation appear essentially conserved throughout eukaryotes [1,2]
Our results demonstrate that human RIF1-phosphatase 1 (PP1) plays a dual role in replication control—having a repressive role at the step of origin activation, as well as a positive function in supporting origin licensing that may be specific to human cells
The constructs were integrated at the FRT site of the Flp-In T-REx 293 human cell line, creating a set of stable cell lines with either wild-type RIF1 or RIF1-pp1bs expressed under a doxycycline-inducible promoter

Summary

Introduction

The process of DNA replication is critical during cell proliferation for daughter cells to inherit a complete set of genomic information.DNA replication is tightly controlled to prevent either under- or over-replication, with the establishment of licensed DNA replication origins and their subsequent activation closely coupled with other cell cycle events.Best understood in budding yeast, the mechanisms controlling origin establishment and activation appear essentially conserved throughout eukaryotes [1,2]. DNA replication is tightly controlled to prevent either under- or over-replication, with the establishment of licensed DNA replication origins and their subsequent activation closely coupled with other cell cycle events. Later in the cell cycle, origin licensing is prevented by cyclin-dependent kinase (CDK) activity, which in human cells inhibits the loading of MCM complex through several mechanisms [4]. One of these is cell cycledependent degradation of ORC1 protein: ORC1 phosphorylation by cyclin A–CDK2 during S/G2 cell cycle phases triggers its proteasomal degradation [5,6]

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