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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

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Inhibition of DNA binding of MCM2-7 complex by phosphorylation with cyclin-dependent kinases

Mariko Moritani1, Yukio Ishimi

  • 1College of Science, Ibaraki University, Mito, Ibaraki 351-8511, Japan.

Journal of Biochemistry
|July 19, 2013
PubMed

Insights

Cyclin-dependent kinases (CDKs) regulate DNA replication by phosphorylating proteins. Phosphorylation of MCM4 by CDK inhibits MCM2-7 DNA binding, preventing re-replication.

Area of Science:

  • Molecular Biology
  • Cell Cycle Regulation
  • DNA Replication

Background:

  • Cyclin-dependent kinases (CDKs) are crucial for cell cycle control, particularly in preventing DNA re-replication.
  • Phosphorylation of replication proteins by CDKs can inactivate them and affect their function.
  • Previous studies show inverse correlations between MCM4 and RPA2 phosphorylation and their chromatin binding.

Purpose of the Study:

  • To investigate the in vitro phosphorylation of key human DNA replication proteins by various kinases.
  • To determine the effect of CDK-mediated phosphorylation on the DNA-binding abilities of MCM2-7 and RPA.
  • To identify the specific sites on MCM4 responsible for CDK-induced inhibition of DNA binding.

Main Methods:

  • In vitro phosphorylation assays using purified human replication proteins (MCM2-7, RPA, TRESLIN, CDC45, RECQL4) and different CDK complexes (CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB) and other kinases (CHK1, CHK2, CDC7/DBF4).
  • Gel-shift analysis to assess the DNA-binding capabilities of MCM2-7 and RPA before and after phosphorylation.
  • Site-directed mutagenesis of MCM4 to alter serine and threonine residues in the amino-terminal region.

Main Results:

  • MCM4, RPA2, TRESLIN, and RECQL4 were identified as substrates for CDKs.
  • Phosphorylation of RPA by CDK2/cyclinA did not alter its DNA-binding ability.
  • Phosphorylation of MCM4 by CDK2/cyclinA significantly inhibited the DNA-binding ability of the MCM2-7 complex.
  • Mutating six serine/threonine sites in the MCM4 amino-terminus rendered MCM2-7 resistant to CDK-mediated inhibition.

Conclusions:

  • CDK-mediated phosphorylation of MCM4 directly impacts the DNA-binding activity of the MCM2-7 complex.
  • This phosphorylation event is a key mechanism for dislodging MCM2-7 from chromatin or preventing its re-association.
  • These findings elucidate a critical step in preventing DNA re-replication during the cell cycle.
Keywords:
Chemical Modification < EnzymeDNA-Binding < ProteinPhosphorylation < ProteinProtein Kinases/CDKs < Signal TransductionReplication < DNA

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