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DNA Repair pp 225-244 | Cite as

Assembling the Human Resectosome on DNA Curtains

  • Michael M. Soniat
  • Logan R. Myler
  • Ilya J. FinkelsteinEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 1999)

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.

Key words

DNA curtains Homologous recombination Bloom’s syndrome helicase (BLM) DNA nuclease (DNA2) Exonuclease 1 (EXO1) 
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Notes

Acknowledgments

We are indebted to Drs. Mauro Modesti and Tanya Paull for plasmids, cell pellets, and other reagents. This work was supported by the National Institutes of Health (GM120554 and CA092584) and the Welch Foundation (F-l808 to I.J.F.). M.M.S. is supported by a Postdoctoral Fellowship, PF-17-169-01-DMC, from the American Cancer Society. L.R.M. is supported by the National Cancer Institute (CA212452).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Michael M. Soniat
    • 1
  • Logan R. Myler
    • 1
  • Ilya J. Finkelstein
    • 1
    • 2
    Email author
  1. 1.Department of Molecular Biosciences and Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA
  2. 2.Center for Systems and Synthetic BiologyThe University of Texas at AustinAustinUSA

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