INTERACTION OF THE YEAST RAD7 AND SIR3 PROTEINS - IMPLICATIONS FOR DNA-REPAIR AND CHROMATIN STRUCTURE

We have used the two-hybrid system to identify proteins that interact with the product of RAD7, a gene involved in DNA repair. A screen of y east genomic DNA-GAL4 activation domain (GAD) fusion gene library allo wed the isolation of plasmids containing sequences corresponding to th e 3' end of the SIR3 gene. This gene is known to be involved in the pr oduction of transcriptionally silent DNA at the cryptic mating-type ca ssettes and at telemores. The cloned sequences coded for amino acids 3 07-979 of the Sir3 protein. A sir3 deletion allele, constructed in an isogenic rad7-deletion strain, rescued approximately one-quarter of th e UV sensitivity associated with the rad7 deletion, indicating that th e two genes interact genetically. Radiolabeled fusion proteins, made w ith the glutathione S-transferase (GST) gene in the vector pGEX-2T, we re purified from Echerichia coli and shown to interact in vitro. This evidence suggests that the Sir3 protein interacts with the Rad7 protei n allow the nucleotide excision repair complex access to transcription ally inactive chromatin. The proportions of 5-FOA-resistant cells in c ultures from isogenic RAD(+) and rad7-Delta strains containing a telom eric URA3 gene were similar, suggesting that the RAD7 gene is not invo lved in the production or structure of transcriptionally silent chroma tin at the telomeres. RAD7-dependent DNA repair of transcriptionally s ilent chromatin was shown not to induce expression of a telomeric copy of the URA3 gene, suggesting that repair of transcriptionally silent chromatin differs from transcriptionally active chromatin. Expression of a telomeric copy of the URA3 gene was stimulated in a rad7-Delta mu tant, suggesting that repair of lesions in the absence of RAD7 can res ult in the activation of transcriptionally silenced genes.