Abstract
Telomerase adds telomeric repeat sequences to chromosome ends using a short region of its RNA subunit as a template. Telomerase RNA subunits are phylogenetically highly divergent, and different strategies have evolved to demarcate the boundary of the template region. The recent identification of the gene encoding telomerase RNA in the fission yeast Schizosaccharomyces pombe (ter1+) has opened the door for structure-function analyses in a model that shares many features with the telomere maintenance machinery of higher eukaryotes. Here we describe a structural element in TER1 that defines the 5' boundary of the template. Disruption of a predicted long range base pairing interaction between template-adjacent nucleotides and a sequence further upstream resulted in reverse transcription beyond the template region and caused telomere shortening. Normal telomere length was restored by combining complementary nucleotide substitutions in both elements, showing that base pairing, not a specific sequence, limits reverse transcription beyond the template. The template boundary described here resembles that of budding yeasts and some mammalian telomerases. However, unlike any previously characterized boundary element, part of the paired region overlaps with the template itself, thus necessitating disruption of these interactions during most reverse transcription cycles. We show that changes in the paired region directly affect the length of individual telomeric repeat units. Our data further illustrate that marginal alignment of the telomeric 3' end with RNA sequences downstream of the template is responsible for primer slippage, causing incorporation of strings of guanosines at the start of a subset of repeats.
Highlights
Telomerase is comprised of the catalytic protein subunit TERT and a TER component [7]
Mutational analysis demonstrated that the P1 helix and its distance from the template are critical for template boundary definition in human telomerase [21]
In Kluyveromyces and Candida yeast species, perfect copies of up to 26-nucleotide template sequences are repeatedly copied onto chromosome ends
Summary
Constructs and Strains—A knock-out for ter1ϩ was generated by replacing nucleotides 23–1422 of the RNA-encoding region with the kanamycin resistance gene in a diploid strain as described [33]. Mutations in ter were introduced using the QuikChange II XL site-directed mutagenesis kit (Stratagene) on pJW10, a plasmid containing a genomic DNA fragment of the ter1ϩ locus [31]. Spores harboring a mutant ter plasmid were germinated on pombe minimal glutamate (PMG) supplemented with adenine, histidine, and leucine, and haploid strains deleted for the genomic copy of ter1ϩ were identified by growth on YEA geneticin. Radiolabeled primer extension products were separated on 8% Tris borate-EDTA, 8 M urea gels next to dideoxynucleotide chain termination sequencing reactions using cloned ter1ϩ as a template. S. pombe telomeres were cloned from genomic DNA samples using the G overhang capture assay [31]. PCR products were cloned into the pCR4blunt-TOPO vector (Invitrogen) for sequence analysis of individual telomeres. The TweenMotif program is available for download from the Baumann Lab web site, along with source code and sample analysis files
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