Abstract
Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.
Highlights
Telomeres, nucleoprotein complexes located at the ends of eukaryotic chromosomes, protect chromosome ends from degradation, from telomere-telomere fusion events, and from being recognized as double-stranded DNA breaks (Wellinger & Zakian, 2012; de Lange, 2018)
We examined the telomere length of cells that had undergone ~30 population doublings after isolation of the haploid spores and found that, upon loss of telomerase, mutant telomeres became extremely heterogeneous and degraded. 187 Rif1 and Sir4, but not Rif2 nor Sir3, association to tlc1-tm telomeres is decreased Rap1, through its C-terminal domain, recruits Rif1, Rif2, Sir3, and Sir4 to telomeres (Hardy et al, 1992; Wotton & Shore, 1997; Moretti et al, 1994)
We investigated the consequences of depleting Rap1 from S. cerevisiae telomeres
Summary
Nucleoprotein complexes located at the ends of eukaryotic chromosomes, protect chromosome ends from degradation, from telomere-telomere fusion events, and from being recognized as double-stranded DNA breaks (Wellinger & Zakian, 2012; de Lange, 2018). Telomeric DNA consists of tandem G/C-rich repeats of doublestranded DNA with the G-rich strand extending to form a 3′ single-stranded overhang. These repeats are bound by specialized proteins—some to the double-stranded region and others to the 3′ overhang—which are important for proper telomere function. Telomere length is maintained by a dynamic process of shortening and lengthening. Telomeres shorten due to incomplete DNA replication and nucleolytic degradation, and are lengthened by the action of a specialized reverse transcriptase called telomerase (Wellinger, 2014). Telomerase consists of a catalytic protein subunit and an RNA subunit, and extends telomeres by iterative reverse transcription of a short G-rich sequence to the 3′ overhang, using the RNA subunit as a template
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