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 DNA containing damage 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.