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Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex

Ivar Ilves1, Nele Tamberg, Michael R Botchan

  • 1Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

Proceedings of the National Academy of Sciences of the United States of America
|August 3, 2012
PubMed

Insights

DNA damage slows eukaryote chromosome replication. We show the Cdc45/MCM2-7/GINS (CMG) helicase is inhibited by Chk2 phosphorylation, revealing a key mechanism for genomic stability.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic DNA replication forks are targeted by signaling pathways upon DNA damage to ensure genomic inheritance.
  • The precise molecular mechanisms by which DNA damage responses (DDR) are mediated at the replication fork remain incompletely understood.

Purpose of the Study:

  • To investigate the role of protein phosphorylation in regulating the activity of the CMG helicase complex during DNA damage response.
  • To identify the specific components of the CMG complex targeted by DDR kinases and elucidate the functional consequences of these modifications.

Main Methods:

  • In vitro biochemical assays using recombinant Drosophila melanogaster CMG complex.
  • Phosphatase and kinase (Chk1, Chk2) treatments to assess CMG helicase activity.
  • Identification of phosphorylation sites using biochemical methods.
  • Analysis of CMG complex phosphorylation in response to ionizing radiation in developing fly embryos.

Main Results:

  • CMG helicase activity can be inhibited by protein phosphorylation.
  • Phosphatase treatment stimulates CMG activity, while Chk2, but not Chk1, interferes with it in vitro.
  • Chk2 phosphorylation targets MCM subunits 3 and 4, and the GINS protein Psf2.
  • Ionizing radiation induces hyperphosphorylation of Psf2 in the active CMG complex in vivo.

Conclusions:

  • Direct modification of the CMG helicase by Chk2 is a critical nexus in the DNA damage response.
  • Phosphorylation of MCM and GINS subunits by Chk2 allosterically affects CMG helicase function, contributing to replication fork stability.
  • These findings provide molecular insight into how replication is regulated during genotoxic stress.

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