Assembling the Human Resectosome on DNA Curtains.
Methods Mol Biol
; 1999: 225-244, 2019.
Article
in En
| MEDLINE
| ID: mdl-31127580
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
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Microfluidic Analytical Techniques
/
Recombinational DNA Repair
/
Single Molecule Imaging
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2019
Document type:
Article
Affiliation country:
United States