Assembling the human resectosome on DNA curtains

Michael M. Soniat, Logan R. Myler, Ilya J Finkelstein

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages225-244
Number of pages20
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1999
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Fingerprint

DNA
Microfluidics
Double-Stranded DNA Breaks
Homologous Recombination
Maintenance
Genome
Proteins
exodeoxyribonuclease I
Bloom syndrome protein

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Soniat, M. M., Myler, L. R., & Finkelstein, I. J. (2019). Assembling the human resectosome on DNA curtains. In Methods in Molecular Biology (pp. 225-244). (Methods in Molecular Biology; Vol. 1999). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9500-4_14

Assembling the human resectosome on DNA curtains. / Soniat, Michael M.; Myler, Logan R.; Finkelstein, Ilya J.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 225-244 (Methods in Molecular Biology; Vol. 1999).

Research output: Chapter in Book/Report/Conference proceedingChapter

Soniat, MM, Myler, LR & Finkelstein, IJ 2019, Assembling the human resectosome on DNA curtains. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1999, Humana Press Inc., pp. 225-244. https://doi.org/10.1007/978-1-4939-9500-4_14
Soniat MM, Myler LR, Finkelstein IJ. Assembling the human resectosome on DNA curtains. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 225-244. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9500-4_14
Soniat, Michael M. ; Myler, Logan R. ; Finkelstein, Ilya J. / Assembling the human resectosome on DNA curtains. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 225-244 (Methods in Molecular Biology).
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