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Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex

Lena R Kundu1, Yuji Kumata, Naoko Kakusho

  • 1Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

Nucleic Acids Research
|April 28, 2010
PubMed

Insights

Destabilizing Cdc6 (cell division cycle protein 6) from chromatin after origin licensing is crucial for DNA replication initiation. Deregulation of Cdc6 arrests DNA replication by inhibiting Cdc7-mediated Mcm2-7 phosphorylation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Mcm2-7 complex is recruited to DNA replication origins by ORC, Cdc6, and Cdt1, a process termed origin licensing.
  • Cdc6 is essential for origin licensing but is destabilized from chromatin after Mcm2-7 loading.

Purpose of the Study:

  • To investigate the role of Cdc6 deregulation in suppressing DNA replication in Xenopus egg extracts.
  • To elucidate the mechanism by which Cdc6 influences DNA replication initiation.

Main Methods:

  • Xenopus egg extracts were used to study DNA replication.
  • Chromatin immunoprecipitation and Western blotting were employed to analyze protein loading and phosphorylation.
  • In vitro kinase assays were performed using purified proteins.

Main Results:

  • Deregulation of Cdc6 after origin licensing arrests DNA replication specifically after Cdc7 binding, independent of ATM/ATR checkpoints.
  • Cdc6 deregulation suppresses Cdc7-mediated hyperphosphorylation of Mcm4, inhibiting the loading of Cdc45, Sld5, and DNA polymerase α.
  • Cdc6 modulates Mcm2-7 phosphorylation in a chromatin-dependent manner, rather than directly inhibiting Cdc7 kinase activity.

Conclusions:

  • Cdc6 on chromatin acts as a critical modulator of Cdc7-mediated Mcm2-7 phosphorylation.
  • Destabilization of Cdc6 from chromatin following origin licensing is essential for the transition to DNA replication initiation.

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