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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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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