Single-Molecule Imaging Reveals How Mre11-Rad50-Nbs1 Initiates DNA Break Repair

Mol Cell. 2017 Sep 7;67(5):891-898.e4. doi: 10.1016/j.molcel.2017.08.002. Epub 2017 Aug 31.

Abstract

DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.

Keywords: DNA repair; Ku; MRN; double-strand break; resection; resectosome; single-molecule.

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Adducts / genetics
  • DNA Adducts / metabolism
  • DNA Breaks, Double-Stranded*
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Diffusion
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Humans
  • Ku Autoantigen / genetics
  • Ku Autoantigen / metabolism
  • MRE11 Homologue Protein
  • Microscopy, Fluorescence
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Nucleosomes / enzymology*
  • Nucleosomes / genetics
  • Recombinational DNA Repair*
  • Single Molecule Imaging*
  • Time Factors

Substances

  • Cell Cycle Proteins
  • DNA Adducts
  • DNA-Binding Proteins
  • MRE11 protein, human
  • NBN protein, human
  • Nuclear Proteins
  • Nucleosomes
  • EXO1 protein, human
  • Exodeoxyribonucleases
  • MRE11 Homologue Protein
  • Rad50 protein, human
  • XRCC5 protein, human
  • Xrcc6 protein, human
  • Ku Autoantigen
  • DNA Repair Enzymes