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Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention

Claudia Lukas1, Fredrik Melander, Manuel Stucki

  • 1Danish Cancer Society, Institute of Cancer Biology, Copenhagen, Denmark. lukas@biobase.dk

The EMBO Journal
|June 18, 2004
PubMed

Insights

Mediator of DNA damage checkpoint 1 (Mdc1) is crucial for sustained Nbs1 engagement at DNA double-strand breaks (DSBs). Mdc1 acts as an H2AX-dependent platform, switching Nbs1 interactions from transient to sustained binding.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Mediator of DNA damage checkpoint 1 (Mdc1) plays a role in DNA double-strand break (DSB) responses.
  • Mdc1 interacts with the Mre11-Rad50-Nbs1 (MRN) complex, involved in DSB recognition, signaling, and repair.

Purpose of the Study:

  • To investigate the role of Mdc1 in the recruitment and retention of Nbs1 at DSB sites.
  • To elucidate the relationship between Mdc1, Nbs1, H2AX, and ATM in DNA damage response pathways.

Main Methods:

  • Utilized live human cells to observe transient interactions.
  • Employed siRNA to ablate Mdc1 and H2AX.
  • Investigated Nbs1 FHA domain mutations.
  • Monitored phosphorylation of ATM and H2AX.

Main Results:

  • Transient Nbs1 interaction and ATM phosphorylation at DSBs are Mdc1-independent.
  • Mdc1 ablation or Nbs1 FHA domain mutation reduces Nbs1 chromatin affinity and causes dispersal.
  • H2AX phosphorylation is normal in Mdc1-deficient cells, but sustained Nbs1 engagement is impaired.
  • Mdc1, but not Nbs1, is immobilized to chromatin post-DSB.
  • H2AX depletion prevents Mdc1 relocalization and Nbs1 chromatin engagement.

Conclusions:

  • Mdc1 acts as an H2AX-dependent platform for sustained Nbs1 interaction at DSBs.
  • Mdc1 facilitates a switch from transient to sustained Nbs1 binding to DSB-flanking chromatin.
  • This mechanism is critical for efficient DNA damage response and repair.

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