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Rapid DNA double-strand breaks resulting from processing of Cr-DNA cross-links by both MutS dimers

Mindy F Reynolds1, Elizabeth C Peterson-Roth, Ivan A Bespalov

  • 1Department of Pathology, Laboratory Medicine, Brown University, Providence, Rhode Island 02912 , USA.

Cancer Research
|January 15, 2009
PubMed

Insights

Mismatch repair (MMR) proteins, MutSalpha and MutSbeta, cooperate to cause DNA double-strand breaks (DSB) from chromium(VI) damage. This MMR-dependent DSB induction leads to cell death and chromosomal breakage.

Area of Science:

  • Molecular Biology
  • Genotoxicology
  • DNA Repair

Background:

  • Mismatch repair (MMR) enhances the toxicity of chemotherapeutic agents and carcinogens.
  • DNA double-strand breaks (DSB) are crucial in MMR-dependent cell death induced by DNA alkylating drugs.
  • Carcinogenic chromium(VI) rapidly induces DSB through mechanisms not fully understood.

Purpose of the Study:

  • To investigate DNA damage detection mechanisms by MMR proteins in response to chromium(VI).
  • To elucidate the role of MMR proteins, specifically MutSalpha (MSH2-MSH6) and MutSbeta (MSH2-MSH3), in chromium-induced DSB formation.
  • To understand the sequential recruitment and cooperation of MMR pathways in processing chromium-DNA cross-links.

Main Methods:

  • Binding assays using DNA probes with ascorbate-Cr-DNA and cysteine-Cr-DNA cross-links.
  • Cellular assays measuring DSB, micronuclei, and apoptosis in human cells treated with chromate.
  • In situ fractionation to identify MSH6 and MSH3 chromatin foci formation.
  • Inhibition studies using aphidicolin to block DNA synthesis.
  • Chromosome transfer experiments into MSH3-null cells.

Main Results:

  • MSH2-MSH6 (MutSalpha) bound effectively to specific Cr-DNA cross-links, but not to binary Cr-DNA adducts.
  • MSH2-MSH3 (MutSbeta) was essential for DSB, micronuclei, and apoptosis induction by chromate, despite not being involved in initial damage recognition.
  • MSH6 and MSH3 chromatin foci formed in late S phase, dependent on MSH6 and MLH1 for MSH3 foci.
  • DSB production correlated with S to G2 phase progression and was blocked by aphidicolin.
  • Alternative MSH3-like activity restored chromate sensitivity in MSH3-null cells.

Conclusions:

  • Sequential recruitment and cooperation between MutSalpha and MutSbeta are critical for DSB and chromosomal breakage induced by chromium(VI).
  • MMR-dependent processing of Cr-DNA cross-links is the primary mechanism for DSB induction at low to moderate chromium(VI) doses.
  • The findings reveal a novel mechanism of MMR involvement in genotoxicity mediated by chromium compounds.

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