Analysis of DNA repair and chromatin assembly in vitro using immobilized damaged DNA substrates.
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.
Mello
Jill A
JA
Research Section, Institut Curie, UMR218 du Centre National de la Recherche Scientifique, Paris, France.
Moggs
Jonathan G
JG
Almouzni
Geneviève
G
D002474:Cell-Free System; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008280:Magnetics
D002843:Chromatin; D004268:DNA-Binding Proteins