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Updated: Aug 23, 2026

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Analysis of DNA repair and chromatin assembly in vitro using immobilized damaged DNA substrates
Jill A Mello1, Jonathan G Moggs, Geneviève Almouzni
1Research Section, Institut Curie, UMR218 du Centre National de la Recherche Scientifique, Paris, France.
Abstract:
Significant advances have been made in identifying a complex network of proteins that could play a role in the repair of DNA damage in the context of chromatin. Insights into this process have been obtained by combining damaged DNA substrates with mammalian cell-free systems that contain both DNA repair and chromatin assembly activities. The methods described in this chapter provide a powerful approach for the detection of proteins recruited during the recognition and repair of DNA lesions, including repair proteins and chromatin-associated factors. Substrates for the recruitment assay consist of damaged DNA that is immobilized on magnetic beads. A human cell-free system that supports both DNA repair and chromatin assembly is incubated with the immobilized DNA-damaged substrates, and proteins associated with the DNA are then isolated and subjected to analysis. We present here protocols for preparing bead-linked DNA substrates containing different types of lesions, for the reaction of the damaged DNA with cell-free systems, and for the subsequent analysis of proteins that are recruited to the immobilized damaged DNA substrates.
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