Telomere fusions caused by mutating the terminal region of telomeric DNA.

Mutations in the template region of a telomerase RNA gene can lead to the corresponding sequence alterations appearing in newly synthesized telomeric repeats. We analyzed a set of mutations in the template region of the telomerase RNA gene (TER1) of the budding yeast Kluyveromyces lactis that were predicted to lead to synthesis of mutant telomeric repeats with disrupted binding of the telomeric protein Rap1p. We showed previously that mutating the left side of the 12-bp consensus Rap1p binding site led to immediate and severe telomere elongation. Here, we show that, in contrast, mutating either the right side of the site or both sides together leads initially to telomere shortening. On additional passaging, certain mutants of both categories exhibit telomere-telomere fusions. Often, six new Bal-31-resistant, telomere repeat-containing bands appeared, and we infer that each of the six K. lactis chromosomes became circularized. These fusions were not stable, appearing occasionally to resolve and then reform. We demonstrate directly that a linear minichromosome introduced into one of the fusion mutant strains circularized by means of end-to-end fusions of the mutant repeat tracts. In contrast to the chromosomal circularization reported previously in Schizosaccharomyces pombe mutants defective in telomere maintenance, the K. lactis telomere fusions retained their telomeric DNA repeat sequences.

Template boundary in a yeast telomerase specified by RNA structure.

The telomerase ribonucleoprotein has a phylogenetically divergent RNA subunit, which contains a short template for telomeric DNA synthesis. To understand how telomerase RNA participates in mechanistic aspects of telomere synthesis, we studied a conserved secondary structure adjacent to the template. Disruption of this structure caused DNA synthesis to proceed beyond the normal template boundary, resulting in altered telomere sequences, telomere shortening, and cellular growth defects. Compensatory mutations restored normal telomerase function. Thus, the RNA structure, rather than its sequence, specifies the template boundary. This study reveals a specific function for an RNA structure in the enzymatic action of telomerase.

Molecular manifestations and molecular determinants of telomere capping.

Multiple interacting components of the telomere, together with telomerase (and sometimes recombination), determine whether a telomere will be functional, allowing cell proliferation. The various components reinforce each other, providing for a robust and resilient system of protection and replenishment of telomeres. A characteristic of a telomere is that its structural features elicit responses that allow it to be dynamically recapped. Eliciting a DNA damage response through uncapping of a telomere appears to be one way in which telomerase action at that telomere is stimulated. Thus, as long as a timely and appropriate recapping of the telomere is possible, regulated uncapping of a telomere appears to be not only normal, but even required for optimal telomere maintenance. Telomere length and the presence of telomerase provide an example of a pair of interacting components that determine telomere capping function. Telomerase is dispensable in cells with sufficiently long telomeres; but in cells with short telomeres lacking telomerase, cells lose the ability to proliferate, and in some cell types, telomere fusions are increased. However, expressing telomerase can make even very short telomeres functional. Many interesting questions remain as to the mechanisms of these biological effects.

Specific telomerase RNA residues distant from the template are essential for telomerase function.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Identification of Kluyveromyces lactis telomerase: discontinuous synthesis along the 30-nucleotide-long templating domain.

Telomeres in the budding yeast Kluyveromyces lactis consist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactis telomerase RNA, encoded by the TER1 gene, is approximately 1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactis whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactis telomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.