Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant. The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint. Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants. These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.     

Dynamics of telomeric DNA turnover in yeast.

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.     

Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells leads to altered telomeres that correlate with reduced cell viability.

Telomerase synthesizes telomeric DNA by copying the template sequence of its own RNA component. In Tetrahymena thermophila and yeast (G. Yu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature 344:126-131, 1990; M. McEachern and E. H. Blackburn, Nature 376:403-409, 1995), mutations in the template domain of this RNA result in synthesis of mutant telomeres and in impaired cell growth and survival. We have investigated whether mutant telomerase affects the proliferative potential and viability of immortal human cells. Plasmids encoding mutant or wild-type template RNAs (hTRs) of human telomerase and the neomycin resistance gene were transfected into human cells to generate stable transformants. Expression of mutant hTR resulted in the appearance of mutant telomerase activity and in the synthesis of mutant telomeres. Transformed cells were not visibly affected in their growth and viability when grown as mass populations. However, a reduction in plating efficiency and growth rate and an increase in the number of senescent cells were detected in populations with mutant telomeres by colony-forming assays. These results suggest that the presence of mutant telomerase, even if coexpressed with the wild-type enzyme, can be deleterious to cells, likely as a result of the impaired function of hybrid telomeres.