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The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for single-stranded DNA extrusion and helicase assembly

Irina Bruck1, Daniel L Kaplan2

  • 1From the Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida 32306.

The Journal of Biological Chemistry
|December 5, 2014
PubMed

Insights

Budding yeast Dbf4-Cdc7 kinase phosphorylates Mcm2 during S phase, impacting DNA replication. This phosphorylation is crucial for CMG helicase assembly and function.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The eukaryotic CMG helicase (Cdc45, Mcm2-7, GINS) is essential for DNA replication.
  • The Dbf4-Cdc7 kinase (DDK) is known to phosphorylate Mcm2 in vitro, but its in vivo role remains unclear.

Purpose of the Study:

  • To investigate the in vivo function of Dbf4-Cdc7 kinase phosphorylation of Mcm2 in budding yeast.
  • To elucidate the mechanism by which DDK phosphorylation affects CMG helicase assembly and DNA replication.

Main Methods:

  • In vivo phosphorylation assays in budding yeast.
  • Analysis of DNA replication phenotypes, including growth defects and origin firing.
  • Biochemical assays to assess protein-protein interactions and Mcm2-7 ring opening.

Main Results:

  • Dbf4-Cdc7 phosphorylates Mcm2 in vivo during S phase, and inhibiting this phosphorylation causes severe growth defects.
  • Impaired DDK phosphorylation of Mcm2 leads to reduced DNA replication, decreased single-stranded DNA formation at origins, and disrupted GINS-Mcm2-7 interaction.
  • In vitro, DDK phosphorylation of Mcm2 weakens Mcm2-Mcm5 interaction and promotes Mcm2-7 ring opening, facilitating ssDNA extrusion and GINS assembly.

Conclusions:

  • Dbf4-Cdc7 phosphorylation of Mcm2 is a critical regulatory step in DNA replication.
  • This phosphorylation event likely opens the Mcm2-7 ring, enabling ssDNA extrusion and subsequent GINS loading, thereby facilitating CMG helicase function.
Keywords:
DNA HelicaseDNA ReplicationDNA-binding ProteinInitiationKinasePhosphorylationProtein-Protein Interaction

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