Reversal of DDK-mediated MCM phosphorylation by Rif1-PP1 regulates replication initiation and replisome stability independently of ATR/Chk1

Robert C. Alver, Gaganmeet Singh Chadha, Peter J. Gillespie, J. Julian Blow (Lead / Corresponding author)

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    81 Citations (Scopus)
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    Abstract

    Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNAbound Mcm4 but not phosphorylation of Mcm2 correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation.
    Original languageEnglish
    Pages (from-to)2508-2520
    Number of pages13
    JournalCell Reports
    Volume18
    Issue number10
    Early online date7 Mar 2017
    DOIs
    Publication statusPublished - 7 Mar 2017

    Keywords

    • Cdc7
    • Rif1
    • MCM
    • Xenopus egg cell-free system
    • human tissue culture
    • DNA replication
    • checkpoint signalling
    • DNA damage

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