Assembling the Human Resectosome on DNA Curtains

Methods Mol Biol. 2019;1999:225-244. doi: 10.1007/978-1-4939-9500-4_14.

Abstract

DNA double-strand breaks (DSBs) are a potentially lethal DNA lesions that disrupt both the physical and genetic continuity of the DNA duplex. Homologous recombination (HR) is a universally conserved genome maintenance pathway that initiates via nucleolytic processing of the broken DNA ends (resection). Eukaryotic DNA resection is catalyzed by the resectosome-a multicomponent molecular machine consisting of the nucleases DNA2 or Exonuclease 1 (EXO1), Bloom's helicase (BLM), the MRE11-RAD50-NBS1 (MRN) complex, and additional regulatory factors. Here, we describe methods for purification and single-molecule imaging and analysis of EXO1, DNA2, and BLM. We also describe how to adapt resection assays to the high-throughput single-molecule DNA curtain assay. By organizing hundreds of individual molecules on the surface of a microfluidic flowcell, DNA curtains visualize protein complexes with the required spatial and temporal resolution to resolve the molecular choreography during critical DNA-processing reactions.

Keywords: Bloom’s syndrome helicase (BLM); DNA curtains; DNA nuclease (DNA2); Exonuclease 1 (EXO1); Homologous recombination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Breaks, Double-Stranded
  • DNA Helicases / analysis
  • DNA Helicases / genetics
  • DNA Helicases / isolation & purification
  • DNA Repair Enzymes / analysis
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / isolation & purification
  • Exodeoxyribonucleases / analysis
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / isolation & purification
  • Microfluidic Analytical Techniques / methods*
  • Microscopy, Fluorescence / methods
  • Quantum Dots / chemistry
  • RecQ Helicases / genetics
  • RecQ Helicases / isolation & purification
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinational DNA Repair*
  • Single Molecule Imaging / methods*

Substances

  • Recombinant Proteins
  • EXO1 protein, human
  • Exodeoxyribonucleases
  • Bloom syndrome protein
  • DNA Helicases
  • DNA2 protein, human
  • RecQ Helicases
  • DNA Repair Enzymes