RECONSTITUTION OF YEAST NUCLEOTIDE EXCISION-REPAIR WITH PURIFIED RAD PROTEINS, REPLICATION PROTEIN-A, AND TRANSCRIPTION FACTOR TFIIH

Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from ye ast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purifi ed protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad 2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RN A polymerase II transcription factor TFIIH were purified to near homeg eneity from yeast. We show that these protein factors are both necessa ry and sufficient for dual incision of DNA damaged by either ultraviol et light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstit uted system requires ATP, which cannot be substituted by adenosine 5'- O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indis pensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.