Sn. Guzder et al., RECONSTITUTION OF YEAST NUCLEOTIDE EXCISION-REPAIR WITH PURIFIED RAD PROTEINS, REPLICATION PROTEIN-A, AND TRANSCRIPTION FACTOR TFIIH, The Journal of biological chemistry, 270(22), 1995, pp. 12973-12976
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.