ASSESSING THE REQUIREMENTS FOR NUCLEOTIDE EXCISION-REPAIR PROTEINS OFSACCHAROMYCES-CEREVISIAE IN AN IN-VITRO SYSTEM

Nucleotide excision repair (NER) is the primary mechanism by which bot h Saccharomyces cerevisiae and human cells remove the DNA lesions caus ed by ultraviolet light and other mutagens. This complex process invol ves the coordinated actions of more than 20 polypeptides. To facilitat e biochemical studies of NER in yeast, we have established a simple pr otocol for preparing whole cell extracts which perform NER in vitro, A s expected, this assay of in vitro repair was dependent on the product s of RAD genes such as RAD14, RAD4 and RAD2. Interestingly, it was als o dependent upon proteins encoded by the RAD7, RAD16, and RAD23 genes whose precise roles in NER are uncertain, but not the RAD26 gene whose product is believed to participate in coupling NER to transcription, Replication protein A (RPA/Rpa), known to be required for NER in human cell extracts, was also shown by antibody inhibition and immunodeplet ion experiments to be required for NER in our yeast cell extracts. Mor eover, yeast cells with temperature sensitive mutations in the RFA2 ge ne, which encodes the 34-kDa subunit of Rpa, had increased sensitivity to UV and yielded extracts defective in NER in vitro, These data indi cate that Rpa is an essential component of the NER machinery in S. cer evisiae as it is in mammalian cells.