SIR PROTEINS, RIF PROTEINS, AND CDC13P BIND SACCHAROMYCES TELOMERES IN-VIVO

Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indire ctly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that sis proteins that affect telomere structure or function but which had not been shown previously to bind telomeres in vivo are indeed telomere binding proteins. A promoter-defective all ele of HIS3 was placed adjacent to a chromosomal telomere. Candidate p roteins fused to a transcriptional activation domain were tested for t he ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir3p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins a ctivated the same reporter gene when it was at an internal site on the chromosome. Moreover, Cdc13p did not activate the reporter gene when it was adjacent to an internal tract of telomeric sequence, indicating that Cdc13p binding was telomere limited in vivo. The amino-terminal 20% of Cdc13p was sufficient to target Cdc13p to a telomere, suggestin g that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir-4p, and Cdc13p activated the telomeric report er gene in a strain lacking Sir3p, which is essential for telomere pos ition effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The d ata support models in which the telomere acts as an initiation site fo r TPE by recruiting silencing proteins to the chromosome end.