Increased association of telomerase with short telomeres in yeast

Abstract

Telomere function is mediated by the assembly of a protein complex on an array of telomeric DNA (TG) repeats synthesized by the telomerase enzyme. Telomerase action at chromosome ends is finely tuned by the telomeric complex so that a constant average number of repeats is maintained. This is achieved through a negative feedback process that is sensitive to TG tract length, but whose underlying mechanism is unknown. We show that short telomeres, which are preferential substrates for telomerase, display increased association with the enzyme in the S phase of the cell cycle, when telomerase acts. In addition, we provide support for a molecular mechanism by which this key step of telomerase recruitment is regulated by TG tract length.

Figure 1.

Molecular models leading to preferential elongation of shortened telomeres in yeast. (Top panels) Schematic representation of the association of selected telomeric proteins involved in the regulation of telomerase action with short and normal-length telomeres. Three different hypothetical models pertaining to possible mechanisms regulating the preferential activation of telomerase at short telomeres are illustrated. The regulated interaction between the Cdc13 and Est1 proteins is indicated by a double arrow (“activation” events are indicated throughout by simple green arrows, “repressive” events are indicated by red bars). In the bottom panels, the predicted enrichment at short versus normal-length telomeres for Cdc13, Est1, and Est2 is given.

Figure 2.

A system for the generation of a single shortened telomere on chr V-R. Schematic of the right telomere of chr V-R, generated by homologous recombination at the YER188W locus with a plasmid-borne cassette containing the indicated elements. The positions of the primers used for real-time PCR are shown.

Figure 3.

Preferential recruitment of Est2, Est1, and Tel1, but not Cdc13, to a shortened telomere on chr V-R. Quantification of the association of Myc-tagged Est2 (A), Est1 (B), and Cdc13 (C), and HA-tagged Tel1 (D) with the recombined shortened (right), or unshortened (left) telomere at chr V-R (in red), and with the endogenous telomere on chr VI-R (in blue) by ChIP, on strains released synchronously from a block in G1 (A–C) or dividing asynchronously (D). For A–C, quantification of the S-phase peak signal for the shortened versus unshortened V-R telomere, normalized for the signal obtained at the reference VI-R telomere, gave a value of 3.17 for Est2 (1.69, 2.14, and 1.68 for three independent experiments; P = 0.044), 2.04 for Est1 (2.19 and 2.59 for two replicates; P = 0.009), and 1.07 for Cdc13 (0.86 and 1.06, for two replicates; P = 0.886). Histograms in D represent averages and standard deviations of four independent asynchronously growing cultures (the white columns report association to the nontelomeric PDI1 locus). Student t-tests for V-R and VI-R, V-R and PDI1, and VI-R and PDI1 yielded P values of 0.677, 0.616, and 0.177, respectively, for the strain with the unshortened telomeres, and 0.001, 0.001, and 0.122, respectively, for the strain with the shortened telomeres.

Figure 4.

Preferential recruitment of Est1, but not Cdc13, to a shortened telomere on chr VII-L. Quantification of the association of Myc-tagged Est1 (A) and Cdc13 (B) with the recombined shortened (right) or unshortened (left) telomere at chr VII-L (in green), and with the endogenous telomere on chr VI-R (in blue) by ChIP, on strains released synchronously from a block in G1. Quantification of the S-phase peak signal for the shortened versus unshortened VII-L telomere, normalized for the signal obtained at the reference VI-R telomere, gave a value of 2.04 for Est1 (and 1.93 for an independent experiment) and 1.00 for Cdc13 (and 1.27 for a replicate). In all of these strains, the region between coordinates 16308 and 18192 on chr VII, which contains a putative origin of DNA replication responsible for early replication of shortened telomeres, was deleted.

Figure 5.

A model for a role of the Tel1 kinase in the length-dependent modulation of telomerase association with yeast telomeres. Schematic representation of the proposed model for Tel1-dependent regulation of the preferential association of telomerase (and Est1) to short telomeres (see main text for details).