Methods for studying chromatin assembly coupled to DNA repair.

In the eukaryotic nucleus, the DNA repair machinery operates on chromatin-embedded DNA substrates. Currently, a favored model for DNA repair into chromatin involves the transient disruption of chromatin organization to facilitate access of the repair machinery to DNA lesions. Importantly, this model implies that, in addition to DNA repair, a subsequent step is necessary to restore a proper chromatin structure. To study this latter step, we describe here methods for simultaneously analyzing chromatin assembly and DNA repair both in vitro and in vivo. Several cell-free systems have been developed that reproduce both DNA repair and nucleosome assembly. These in vitro systems are based on the use of defined damaged DNA. Two complementary assays are routinely used: (i) with circular DNA molecules, one can monitor in a combined analysis both repair synthesis and plasmid supercoiling; (ii) with immobilized damaged DNA, one follows specific protein interactions including histone deposition. In addition, in vivo assays have been designed to monitor the recruitment of chromatin assembly factors onto damaged chromatin either at a global level over the whole cell nucleus or locally at sites of DNA damage. Combination of these approaches provides powerful tools to gain insights into the mechanism by which chromatin organization can be restored after repair of DNA lesions.