Yeast telomerase appears to frequently copy the entire template in vivo

Abstract

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG1-3sequence for the most internal ∼100 bp of the 250-350 bp TG1-3repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG1-3sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70Δ and tel1Δ mutants, whose entire telomeres are only ∼100 bp, were examined by analyzing 5.1 kb of cloned TG1-3sequences from telomeres formed during transformation of wild type, yku70Δ and tel1Δ cells. The internal domains were 97-137 bp in wild type cells, 27-36 bp in yku70Δ cells and 7-9 bp in tel1Δ cells. These data suggest that the majority of the tel1Δ cell TG1-3repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70Δ cell telomeres are protected. TG1-3sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG1-3repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG1-3repeats, only those consistent with telomere hybridization to the ACACAC in the 3′-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40-60% of the TG1-3sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3′-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG1-3sequences that mimic this process.