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Structural mechanism of ATP-dependent DNA binding and DNA end bridging by eukaryotic Rad50

Florian Ulrich Seifert1, Katja Lammens1, Gabriele Stoehr1

  • 1Department of Biochemistry and Gene Center, Ludwig-Maximilians-University, Munich, Germany.

The EMBO Journal
|February 21, 2016
PubMed

Insights

The Mre11-Rad50-Nbs1 complex repairs DNA double-strand breaks. Structural studies reveal how Rad50 binds DNA, offering insights into DNA repair mechanisms and the Rad50/SMC/RecN protein family.

Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • The Mre11-Rad50-Nbs1 (MRN) complex is crucial for DNA double-strand break (DSB) repair.
  • The precise ATP-dependent mechanisms of MRN in DNA end processing remain incompletely understood.

Purpose of the Study:

  • To elucidate the structural basis of DNA binding and processing by the eukaryotic Mre11-Rad50 complex.
  • To provide a framework for understanding the role of Rad50 in DNA repair pathways.

Main Methods:

  • X-ray crystallography of Rad50 nucleotide-binding domain (NBD) in complex with DNA or Mre11 Rad50-binding domain (RBD).
  • Small-angle X-ray scattering (SAXS) and cross-linking studies.
  • In vitro DNA binding experiments and in vivo mutational analyses in Saccharomyces cerevisiae.

Main Results:

  • Crystal structures reveal an ATP-dependent Rad50 dimer binding approximately 18 base pairs of DNA.
  • The Rad50 dimer can bridge two DNA ends, showing a preference for 3' overhangs.
  • Structural and functional data were validated through biochemical and genetic approaches.

Conclusions:

  • A structural framework for the eukaryotic Mre11-Rad50 complex architecture is provided.
  • The findings offer insights into the ATP-dependent DNA binding and bridging activities of Rad50.
  • Results may generalize to the DNA interaction mechanisms of the broader Rad50/SMC/RecN protein superfamily.
Keywords:
DNA repairX‐ray crystallographyhomologous recombinationmicrohomology‐mediated end joiningprotein DNA complex

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