More than 35 genes are known to affect
yeast telomeres. We describe a one-hybrid system that provides a novel
in vivo assay to determine which of these genes encode telomere
binding proteins. A reporter gene was placed adjacent to a functional
chromosomal telomere. Candidate proteins fused to a transcriptional
activation domain were tested for their ability to activate
transcription of the telomere-linked gene. Because the reporter gene was
next to a chromosomal telomere, a positive signal is likely to be
biologically relevant. Using this system, Rap1p, Rif1p, Rif2p, Sir2p,
Sir3p, Sir4p and Cdc13p were found to be in vivo telomere binding
proteins. These proteins did not activate the same reporter gene
positioned 20 kb from a telomere. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p
also activated transcription in a sir3 strain, in which others
have shown that there is no telomeric silencing and no association of
the Sir proteins with subtelomeric regions. Cdc13p did not activate a
reporter gene next to an internal tract of telomeric DNA, indicating
that its binding was telomere limited. The one-hybrid system is
versatile; it can identify proteins that associate with telomeres via
protein-protein interactions or by direct binding to either duplex or
single-stranded telomeric DNA and distinguish telomere-limited binding
proteins from those that also bind internal tracts of telomeric
sequence.
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