Abstract
The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.
Highlights
Telomeres, the physical ends of linear chromosomes, are required for the complete replication and stable maintenance of eukaryotic chromosomes
We demonstrate that Pif1p abundance is cell cycle regulated with its highest expression at the same time when telomerase acts
Pif1p Expression Is Cell Cycle Regulated To determine if Pif1p levels vary during the cell cycle, wildtype cells were arrested in late G1 phase with alpha factor and released synchronously into the cell cycle
Summary
The physical ends of linear chromosomes, are required for the complete replication and stable maintenance of eukaryotic chromosomes. They are comprised of stretches of repeated C/G-rich DNA with the G-rich strand extended to form a 39 single-stranded tail (G-tail). The essential protein Cdc13p is a telosome component that binds the single-stranded G-tail and is required to protect telomeric DNA from degradation (reviewed in [4,5]). When yeast cells are limited for Cdc13p or for the Ku complex, they have long single-stranded G-tails [6,10]. Cdc13p and Ku are both required for telomere integrity In their absence, telomeres are more prone to C-strand degradation mediated by Exo1p and perhaps other nucleases
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