Nucleotide Excision Repair in Saccharomyces cerevisiae Whole-Cell Extracts

Nucleotide excision repair (NER) is a particularly versatile pathway of DNA repair capable of removing a broad spectrum of DNA lesions in both prokaryotes and eukaryotes (1 –3 ). NER involves steps of damage recognition, incision and excision of the lesion and its flanking DNA, and repair DNA synthesis to fill in the resulting single-stranded gap. Here we describe the techniques used to prepare extracts from Saccharomyces cerevisiae cells capable of performing NER reactions and the details of this in vitro NER assay. Studies of DNA repair in S. cerevisiae have the advantage of being amenable to powerful genetic analyses within a completely sequenced yeast genome. In fact, most of the earlier work on NER in yeast relied on the genetic analyses of rad mutants. On the other hand, the potential for biochemical analysis of NER in yeast has not yet been fully realized owing in part to the lack of a simple in vitro repair system. This is in contrast to the situation in human cells in which the in vitro system developed by Wood and his colleagues (4 ; see Chapter 29 ) has proven instrumental in dissecting the human pathway of NER.