Academic journal article Genetics

TEN1 Is Essential for CDC13-Mediated Telomere Capping

Academic journal article Genetics

TEN1 Is Essential for CDC13-Mediated Telomere Capping

Article excerpt

ABSTRACT

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

GENOME stability is critically dependent upon functional telomeres. DNA ends that lack telomeres, or that have dysfunctional telomeres, are metabolized by DNA repair processes; without an appropriate repair template, such chromosome ends can be resected or joined inappropriately with other chromosome ends. Thus, genomic integrity can be significantly compromised by telomere dysfunction, particularly in proliferating cells where cycles of instability may ensue due to creation of dicentric chromosomes (Bailey and Murnane 2006). Protein complexes that bind to the duplex and single-stranded telomere repeats are key for stabilizing the chromosome ends (de Lange 2005). In proliferating cells, this job is complicated not only because the terminal chromatin must be opened during the process of chromosome replication, but also because additional processes that metabolize DNA ends are active. For example, while nonhomologous end joining processes are preferentially used in repair of DNA double-strand breaks in G1, homologous recombination is preferentially used for this repair in S and G2 (Ferreira and Cooper 2004; Zierhut and Diffley 2008). Given these complexities, it is not surprising that our molecular understanding of how telomere proteins protect chromosomes ends is incomplete.

Budding yeast has been useful for dissecting how cells correctly metabolize their chromosome ends. In Saccharomyces cerevisiae, the terminal DNA comprises approximately 300 bp of TG1-3/C1-3A sequences, ending with a short single-stranded overhang of the G-rich repeats. This 39 overhang is ~12-14 nucleotides, although during the late S/G2 phase of the cell cycle, it becomes longer, >30 nucleotides in length (Wellinger et al. 1993b; Dionne and Wellinger 1996; Larrivee et al. 2004). Central among factors that prevent inappropriate telomere degradation in S. cerevisiae is Cdc13, a protein that binds to single-stranded telomere G-rich repeats (Garvik et al. 1995; Lin and Zakian 1996; Nugent et al. 1996). Reducing Cdc13 function through either the cdc13-1 temperature sensitive (ts) allele or the cdc13-td conditional null (degron) allele results in telomere C-strand loss, with degradation continuing into the subtelomeric chromosomal regions (Garvik et al. …

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