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DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts

Matthew P Stokes1, Ruth Van Hatten, Howard D Lindsay

  • 1The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. matt@mcb.harvard.edu

The Journal of Cell Biology
|September 6, 2002
PubMed

Insights

Methyl methanesulfonate (MMS) triggers DNA damage checkpoints, but the sensing mechanism remains unclear. This study reveals that DNA replication forks are essential for activating these crucial cellular checkpoints.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Genetics

Background:

  • Alkylating agents like methyl methanesulfonate (MMS) induce DNA damage and activate cellular checkpoints.
  • While checkpoint proteins are known, the precise mechanism of damage sensing and checkpoint activation is not fully understood.

Purpose of the Study:

  • To elucidate the mechanism of DNA damage sensing and checkpoint activation in response to alkylation damage.
  • To investigate the role of replication in MMS-induced checkpoint activation.

Main Methods:

  • Reconstitution of the DNA damage checkpoint response to MMS in Xenopus egg extracts.
  • Monitoring of four key indicators: mitotic entry delay, DNA replication slowing, Chk1 phosphorylation, and Rad17 protein association with damaged DNA.

Main Results:

  • MMS-induced checkpoint activation requires entry into S phase.
  • Replication of damaged double-stranded DNA, not single-stranded DNA, triggers checkpoint activation.
  • Replication forks are identified as a critical intermediate in this process.

Conclusions:

  • Replication forks are obligate intermediates in the activation of the DNA damage checkpoint.
  • This finding clarifies a fundamental aspect of DNA damage response pathways.

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