Telomere Extension in Yeast
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Telomere Extension in Yeast
Telomeres are specialized nucleoprotein structures found at the ends of linear eukaryotic chromosomes. Telomeres confer stability to chromosomes by preventing nucleolytic degradation and recombination. They also function in chromosomal localization, nuclear architecture, and repression of nearby genes. The telomeric DNA of most organisms consists of simple tandem repeats that are rich in dG and dT residues on the 3 end-containing strand. This strand is synthesized by a ribonucleoprotein complex called telomerase, an enzyme that is critical to the maintenance of telomere length and function (Ref.1).

The telomeres of the budding yeast Saccharomyces cerevisiae contain ~300 base pairs of double-stranded (TG1-3/C1-3A) sequences. Telomerase recruitment is mediated by a direct protein-protein interaction between CDC13 and the telomerase-associated EST1 and EST2 protein. In the absence of other components of the holoenzyme, EST1 and EST2 form a complex containing TLC1 (the telomerase RNA). The 3 chromosome terminus is bound to the active site, but EST1 has a single-stranded DNA-binding activity that contributes to the interaction of telomerase with the primer substrate. The telomeric TG1-3/C1-3A DNA forms a complex nonnucleosomal chromatin structure, called the telosome. The major component in yeast telosomes is a double-stranded, sequence-specific DNA-binding protein-RAP-1 complex. The copy number of the RAP-1 complex negatively regulates telomere length by inhibiting access of the telomerase enzyme through sequestration of the chromosome terminus (Ref.2). When telomere length exceeds a certain threshold, the binding of excess RAP-1 molecules shift the equilibrium at the telomere into a ‘closed’ conformation that is refractory to telomere elongation. Since RAP-1 bends DNA, one proposed model for the ‘closed’ telomere conformation is a folded back structure that renders the 3 end inaccessible to telomerase. The action of RAP-1 in effecting regulation is mediated through two proteins, RIF1 (RAP-1 Interacting Factor) and RIF2, which interact with the C-terminus of RAP-1 and also with each other; therefore the folded structure is stabilized through RIF-RIF protein interactions. When the telomere shortens below a critical level, owing to incomplete telomere replication, one or more RAP-1 molecules are lost from the telomere, which then shifts the equilibrium at the terminus towards an ‘open’ structure permissive to telomere addition mediated by DNA Polymerase-Alpha. Whether this end structure forms a large duplex loop, as observed for mammalian telomeres is still not confirmed (Ref.3).

Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing SIR2/3/4 (Silent Information Regulator) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Both yKu and the chromatin-associated RAP-1 and SIR proteins are released from telomeres in a Rad9-dependent response to DNA damage (Ref.4). Silencing at all of these sites also involves the amino terminus of histone H4 and presumably results in a heterochromatic structure over the silenced region.

Telomere elongation by telomerase occurs only in late S-phase and coincides with the timing of telomeric DNA replication. In addition to functioning as protective caps on chromosome ends, telomeres contribute to the faithful completion of DNA replication because of the unique mechanism of telomere synthesis (Ref.5). Some organisms normally rely on telomerase; telomerase-independent mechanisms of telomere maintenance exist. Most cells in S.cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe, which lack the gene for telomerase component die. In both S. cerevisiae and K. lactis, generation of survivors requires Rad52-dependent recombination. In S. cerevisiae the majority of cells that survive in the absence of telomerase activity have multiple tandem copies of the subtelomeric Y element and very short terminal tracts of TG1-3/C1-3A DNA (Type I survivors) (Ref.2). In Saccharomyces cerevisiae mutations in the genes coding for the RNA or the protein subunits of telomerase (TLC1 and EST1-3, respectively) cause a delayed lethal phenotype, which is accompanied by progressive telomere shortening.