Sir proteins, Rif proteins, and Cdc13p bind Saccharomyces telomeres in vivo

Area of Science:

• Molecular Biology
• Yeast Genetics
• Chromosomal Biology

Background:

• Numerous genes influence yeast telomeres, but direct protein-telomere interactions are often uncharacterized.
• Understanding direct telomere binding is crucial for elucidating telomere structure and function.

Purpose of the Study:

• To develop and validate a one-hybrid assay for identifying in vivo telomere binding proteins in yeast.
• To confirm telomere binding for six proteins previously known to affect telomere function.

Main Methods:

• A one-hybrid assay was established using a promoter-defective HIS3 reporter gene adjacent to a chromosomal telomere.
• Candidate proteins fused to a transcriptional activation domain were tested for their ability to activate HIS3 transcription.
• Reporter gene assays were performed at internal chromosomal sites and adjacent to internal telomeric sequences to confirm specificity.

Main Results:

• Six proteins (Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p) were identified as in vivo telomere binding proteins.
• Binding was specific to telomeres, as demonstrated by the lack of reporter gene activation at internal chromosomal locations.
• Cdc13p binding was telomere-limited, and its N-terminal 20% was sufficient for telomere targeting.
• Telomeric association of Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p occurred independently of Sir3p and telomere position effect chromatin features.

Conclusions:

• The one-hybrid assay effectively identifies in vivo telomere binding proteins.
• The study confirms direct telomere binding for six key proteins involved in telomere regulation.
• Results support models where telomeres act as initiation sites for silencing protein recruitment and telomere position effect.