TETHERED SIR3P NUCLEATES SILENCING AT TELOMERES AND INTERNAL LOCI IN SACCHAROMYCES-CEREVISIAE

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telo meric TG(1-3) tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA bin ding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 1 65 amino acids thought to contain sites for Sir3p association. Tetheri ng of LexA-Sir3p adjacent to the telomere is sufficient to restore tel omeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LeXA-Sir3p(N205) gain-of-func tion protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is l imiting in telomeric silencing. In addition, LeXA-Sir3p(N205) hyperrep resses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can i nteract with Rap1p-associated factors at the telomere. We also demonst rate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cia with a short tract of telomeric TG(1-3) sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced i n rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.