Sir Proteins, Rif Proteins, and Cdc13p Bind Saccharomyces Telomeres In Vivo

Abstract

Although a surprisingly large number of genes affect yeast telomeres, in most cases it is not known if their products act directly or indirectly. We describe a one-hybrid assay for telomere binding proteins and use it to establish that six 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 allele of HIS3 was placed adjacent to a chromosomal telomere. Candidate proteins fused to a transcriptional activation domain were tested for the ability to activate transcription of the telomere-linked HIS3 gene. Using this system, Rif1p, Rif2p, Sir2p, Sir3p, Sir4p, and Cdc13p were found to be in vivo telomere binding proteins. None of the proteins activated 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, suggesting that its DNA binding domain was within this portion of the protein. Rap1p, Rif1p, Rif2p, Sir4p, and Cdc13p activated the telomeric reporter gene in a strain lacking Sir3p, which is essential for telomere position effect (TPE). Thus, the telomeric association of these proteins did not require any of the chromatin features necessary for TPE. The data support models in which the telomere acts as an initiation site for TPE by recruiting silencing proteins to the chromosome end.

Saccharomyces Rrm3p, a 5′ to 3′ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA

“…6), the replication fork barrier (RFB) in the rDNA (Ivessa et al 2000), highly transcribed promoters (Ivessa et al 2000), and silent replication origins ( Fig. 5; Ivessa et al 2000), are assembled into large protein-DNA complexes (Wright et al 1992;Santocanale and Diffley 1996;Bourns et al 1998). For example, Rrm3p might be part of a chromatin-remodeling machinery that makes such regions accessible to the conventional DNA replication machinery, or Rrm3p might be required to restart forks that collapse as they attempt to pass through these regions.…”

“…This ∼800-bp C 1-3 A/TG 1-3 tract represses transcription of a nearby URA3 gene, albeit inefficiently compared with telomeres (Stavenhagen and Zakian 1994). Because silencing by internal C 1-3 A/TG 1-3 tracts requires Rap1p and the Sir complex (Stavenhagen and Zakian 1994), these tracts must bind many of the proteins found at telomeres, at least in some cells (see also Bourns et al 1998).…”

“…Saccharomyces has two types of subtelomeric repeats, the YЈ element, which is found in up to four tandem copies on about two-thirds of yeast telomeres, and the X element, which is found on virtually all telomeres (Chan and Tye 1983). The telomeric repeats are assembled into a non-nucleosomal DNA protein complex, the telosome (Wright et al 1992), which contains multiple copies of the C 1-3 A/TG 1-3 -binding Rap1 protein (Conrad et al 1990;Wright et al 1992), as well as Sir proteins, Rif proteins, the single-strand TG 1-3 -DNAbinding Cdc13p (Bourns et al 1998;Tsukamoto et al 2001), and the heterodimeric Ku complex (Gravel et al 1998). In contrast, X and YЈ DNA are assembled into nucleosomes (Wright et al 1992).…”

Telomeric Protein Distributions and Remodeling Through the Cell Cycle inSaccharomyces cerevisiae

“…Rif1-and Rif2-fusion proteins joined to a transcriptional transactivator were experimentally capable of associating with internal tracts of TG [1][2][3] repeat DNA linked to a HIS3 reporter gene (Bourns et al, 1998), but it was not determined whether Rif proteins are present on the internal YЈ telomeric tracts of native telomeres. Herein, we report that Rif1p binding extends many kilobases in from the terminal telomere TG 1-3 tract and closely correlates with the Rap1p and Sir protein binding in these regions.…”

Ten1p promotes the telomeric DNA-binding activity of Cdc13p: implication for its function in telomere length regulation

“…In S. cerevisiae, the 3′ overhang of telomere is bound and protected by an RPA-like trimeric protein complex of Cdc13p/Stn1p/Ten1p [9]. Cdc13p, a 105-kDa protein, binds single-stranded telomeric TG 1-3 DNA specifically both in vitro [10,11] and in vivo [12,13]. The DNA-binding domain of Cdc13p resides in a 187-amino acid fragment from the amino acid 500 to 686 [14][15][16][17][18].…”