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Related Experiment Videos

DNA mismatch repair-dependent response to fluoropyrimidine-generated damage.

Mark Meyers1, Mark W Wagner, Anthony Mazurek

  • 1Department of Radiation Oncology and Case Comprehensive Cancer Center, Laboratory of Molecular Stress Responses, Case Western Reserve University, Cleveland, OH 44106, USA.

The Journal of Biological Chemistry
|December 22, 2004
PubMed
Summary

DNA mismatch repair (MMR) genes MLH1 and MSH2 are crucial for detecting 5-fluorouracil incorporation during DNA replication, triggering cell cycle arrest and death in cancer cells.

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Genetics

Background:

  • Previous studies highlighted the necessity of MLH1 for 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity and G2 arrest in cancer cells.
  • The role of MSH2, another key DNA mismatch repair (MMR) gene, in these cellular responses remained less understood.

Purpose of the Study:

  • To investigate the role of MSH2 in cellular responses to FdUrd, similar to previously observed effects with MLH1.
  • To elucidate the mechanism by which MMR influences FdUrd cytotoxicity and DNA incorporation.

Main Methods:

  • Restoration of MSH2 expression in MSH2-deficient human endometrial carcinoma cells (HEC59) and murine embryonic stem cells.
  • Treatment with FdUrd and assessment of cytotoxicity and G2 arrest.
  • Comparison of cellular responses to FdUrd and the thymidylate synthase inhibitor Tomudex.

Related Experiment Videos

  • Analysis of FdUrd incorporation into DNA using radiolabeled FdUrd and methyl-CpG-binding domain 4 glycosylase assays.
  • Investigation of hMutoma-hMSH6 heterodimer's recognition of FdUrd incorporation via ATPase activation assays.

    • Main Results:

    • Restoring MSH2 expression in MMR-deficient cells restored FdUrd-induced cytotoxicity and G2 arrest, mirroring MLH1's effects.
    • MMR status had minimal impact on cellular responses to Tomudex, suggesting a specific role in FdUrd response.
    • MMR-deficient cells showed higher FdUrd incorporation into DNA compared to MMR-proficient cells.
    • The hMutoma-hMSH6 heterodimer specifically recognized 5-fluorouracil (FU) mispaired with guanine (Gua) in DNA, not adenine.
    • Increased levels of FU:Gua misincorporation were detected in the DNA of MMR-deficient cells.

    Conclusions:

    • DNA mismatch repair specifically detects 5-fluorouracil (FU) misincorporated as FU:Gua during the first round of DNA replication.
    • This detection by MMR, particularly involving the MSH2-MSH6 complex, signals a sustained G2 arrest and induces cell lethality.
    • The findings establish a direct mechanistic link between MMR, FU incorporation, and the cytotoxic effects of FdUrd in cancer cells.