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

Assembling the Human Resectosome on DNA Curtains.

Author information

1
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA.
2
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA. ifinkelstein@cm.utexas.edu.
3
Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX, USA. ifinkelstein@cm.utexas.edu.

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

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