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RNA interference inhibition of Mus81 reduces mitotic recombination in human cells

Veronique Blais1, Hui Gao, Cherilyn A Elwell

  • 1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

Molecular Biology of the Cell
|November 18, 2003
PubMed

Insights

The Mus81-Eme1 complex resolves DNA replication forks and Holliday junctions, crucial for mitotic recombination in human cells. This endonuclease activity is essential for maintaining genomic stability.

Area of Science:

  • DNA repair and recombination
  • Molecular biology
  • Genetics

Background:

  • Mus81 is a conserved endonuclease homologous to XPF.
  • In yeast, Mus81 requires a subunit (Eme1/Mms4) for activity.
  • Human Mus81 interacts with a homolog of fission yeast Eme1.

Purpose of the Study:

  • To investigate the function of the human Mus81-Eme1 complex.
  • To determine the role of Mus81-Eme1 in mitotic recombination.
  • To confirm Mus81-Eme1's ability to resolve DNA structures.

Main Methods:

  • Recombinant Mus81-Eme1 complex expression and purification.
  • In vitro cleavage assays using replication forks, 3' flap, and Holliday junction substrates.
  • RNA interference to down-regulate Mus81 in human somatic cells.
  • Rescue experiments using a bacterial Holliday junction resolvase.

Main Results:

  • Recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro.
  • Mus81 self-associates, and Eme1 self-associates, suggesting multimeric complexes.
  • Down-regulation of Mus81 reduces mitotic recombination in human cells.
  • The recombination defect is rescued by a bacterial Holliday junction resolvase.

Conclusions:

  • Mus81-Eme1 directly cleaves key DNA structures involved in recombination.
  • The Mus81-Eme1 complex plays a significant role in mitotic recombination in higher eukaryotes.
  • Mus81-Eme1 likely functions in vivo to resolve Holliday junctions, ensuring genomic stability.

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