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Identification of MCM4 as a target of the DNA replication block checkpoint system

Yukio Ishimi1, Yuki Komamura-Kohno, Hyun-Ju Kwon

  • 1Biomolecular and Technology Department, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. yukio@libra.ls.m-kagaku.co.jp

The Journal of Biological Chemistry
|April 26, 2003
PubMed

Insights

Researchers identified MCM4 phosphorylation as a key event in the DNA replication checkpoint. This phosphorylation, mediated by ATR-CHK1 and CDK2 kinases, inactivates the MCM helicase complex, halting DNA replication fork progression.

Area of Science:

  • Cellular biology
  • Molecular biology
  • Biochemistry

Background:

  • The DNA replication checkpoint prevents genomic instability by halting replication when DNA damage or stress occurs.
  • Key proteins targeted by the replication checkpoint remain largely unidentified.
  • MCM4 is a subunit of the MCM complex, essential for DNA replication initiation and elongation.

Purpose of the Study:

  • To identify proteins targeted by the DNA replication checkpoint.
  • To investigate the role of MCM4 phosphorylation in regulating DNA replication.
  • To elucidate the kinases involved in MCM4 phosphorylation during replication stress.

Main Methods:

  • HeLa cells were treated with DNA synthesis inhibitors (e.g., hydroxyurea) or UV irradiation.
  • Western blotting with anti-phospho-MCM4 antibodies was used to detect phosphorylation.
  • Kinase activity assays and site-directed mutagenesis were employed to identify kinases and phosphorylation sites.
  • MCM4-6-7 helicase activity was measured in vitro following CDK2 phosphorylation.

Main Results:

  • MCM4 is extensively phosphorylated in response to DNA synthesis inhibitors and UV irradiation.
  • ATR-CHK1 and CDK2 kinases are sequentially involved in hydroxyurea-induced MCM4 phosphorylation.
  • Specific phosphorylation sites on MCM4 were identified.
  • CDK2-mediated phosphorylation of MCM4 negatively regulates the DNA helicase activity of the MCM4-6-7 complex.

Conclusions:

  • MCM4 is a direct target of the DNA replication checkpoint.
  • Phosphorylation of MCM4 by ATR-CHK1 and CDK2 leads to MCM helicase inactivation.
  • This inactivation inhibits DNA replication fork progression, contributing to cell cycle arrest under replication stress.

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