TheRAD7, RAD16, andRAD23Genes ofSaccharomyces cerevisiae: Requirement for Transcription-Independent Nucleotide Excision Repair In Vitro and Interactions between the Gene Products

Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeastSaccharomyces cerevisiae, theRAD7, RAD16, andRAD23genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcrip- tionallyactivegenes.Wehaveusedacell-freesystemtostudytherolesoftheRad7,Rad16,andRad23proteins inNER.Transcription-independentNERofaplasmidsubstratewasdefectiveinrad7,rad16,andrad23mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies withrad4, rad7, rad16, andrad23mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro.OurresultsshowthatRad7,Rad16,andRad23arerequiredfortranscription-independentNERinvitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.