Development and characterization of a new assay to examine telomere-protein interactions in vivo
Bourns, Brenda, 1964-
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An in vivo telomere binding assay using one-hybrid methodology was devised. A reporter gene, lacZ, was integrated immediately adjacent to a chromosomal telomere in Saccharomyces cerevisiae. If a telomere binding protein fused to a transcriptional activation domain were expressed in such a strain, the telomere binding portion should bind the telomere, thus bringing the activation domain within reach of the reporter gene and allowing activation. To test the system, the behavior of Rap1p, which is known to bind telomeres, was analyzed. As expected, Rap1 fused to an activation domain activated lacZ when it was near a telomere, but not when the reporter gene was located internally on a chromosome. These results confirmed the utility of a one-hybrid approach to assay telomere binding in vivo. Three other proteins implicated in telomere biology, Rif1p, Sir4p, and Sir3p tested positive in the system. A more sensitive assay was developed in which a minimally active HIS3 allele was used as a reporter. Each of the fusion proteins that tested positive in the lacZ assay, as well as a Sir2 fusion protein, activated HIS3 when it was located adjacent to a telomere. Additionally, in an even more sensitive telomere one-hybrid assay lacking the repression of genes near telomeres, Cdc13p tested positive for telomere association. These results demonstrated that Rif1p, Sir2p, Sir3p, Sir4p, and Cdc13p associate with a bona fide chromosomal telomere in vivo, and provide a new assay for protein-telomere interaction. When HIS3 plus adjacent telomeric sequence was integrated at an internal position on the chromosome, Rap1, Rif1, Sir2, Sir3, and Sir4 fusion proteins were still associated. In contrast, Cdc13p did not bind the internal telomeric sequence. These results strengthen the idea that Cdc13p binds telomeric sequence preferentially when it is located at chromosomal ends and imply that this assay is able to determine whether a protein has increased affinity for actual telomeric ends as opposed to internal telomeric sequence. Finally, the system was used to screen fusion libraries to obtain more information about proteins associated with chromosomal telomeres, as well as to attempt to identify novel telomere binding proteins.
- Pathology