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Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability

Robert C Alver1, Gaganmeet Singh Chadha1, Peter J Gillespie1

  • 1Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Cell Reports
|March 9, 2017
PubMed

Insights

Dbf4-dependent kinases (DDKs) regulate DNA replication initiation by phosphorylating MCM proteins. Rif1-mediated dephosphorylation balances this, ensuring proper replisome stability and cell cycle control.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA replication initiation is a tightly regulated process essential for cell division.
  • Dbf4-dependent kinases (DDKs) target MCM2-7 proteins, crucial for replication.
  • Understanding the regulation of MCM phosphorylation is key to controlling DNA replication.

Purpose of the Study:

  • To investigate the role of MCM phosphorylation in DNA replication.
  • To elucidate the regulatory mechanisms balancing MCM phosphorylation and dephosphorylation.
  • To determine the impact of Rif1 and Chk1 on replication fidelity and stability.

Main Methods:

  • Utilized Xenopus laevis egg extracts and human cell lines.
  • Employed DDK inhibitors (PHA-767491, XL413) to study phosphorylation.
  • Investigated the function of Rif1 and Chk1 in MCM dephosphorylation and replisome stability.

Main Results:

  • Hyper-phosphorylation of DNA-bound Mcm4, not Mcm2, correlates with DNA replication.
  • DDK-dependent MCM phosphorylation is reversed by Rif1-recruited protein phosphatase 1 (PP1).
  • Loss of Rif1 enhances MCM phosphorylation, increases replication initiation rate, and impairs inhibitor response.
  • Rif1 mediates MCM dephosphorylation at replication forks; replisome stability depends on Chk1.

Conclusions:

  • Replication initiation and replisome stability are regulated by MCM phosphorylation.
  • A balance between DDK-dependent phosphorylation and Rif1-mediated dephosphorylation maintains MCM phosphorylation.
  • This balance is critical for controlling DNA replication initiation and ensuring genomic stability.
Keywords:
Cdc7DNA damageDNA replicationMCMRif1Xenopus egg cell-free systemcheckpoint signalinghuman tissue culture

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