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

Abstract

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.

Figure 1.

Summary of telomere profile and growth phenotypes of 63 mutants. (A) Sequence of the template cassette. The wild-type TLC1 sequence surrounding the template region is shown on top. The template region is in capitals and positions 476–474 are underlined. Mutations that create SphI and SalI sites are in capitals. The SphI and SalI sites are underlined with arrows pointing to the bases where the restriction enzymes cut. Sequence of the Rap1p consensus binding site is shown in 3′-5′ direction underneath. (B) Sequences of the oligos used to construct the template mutant library. (C) Summary of telomere profile and growth phenotypes of 63 mutants. Sequence is shown in 3′-5′ direction. Telomere length for each mutant is characterized as described in text. Cell growth for each mutant is scored as wild type or close to wild-type growth (+++), distinguishably sicker than wild type (++), very sick (+), or senescent (-). Stars indicate mutants that showed immediate slow growth but later recovered. Mutants that were recovered in the screen in Forstemann et al. (2003) are checked. Mutants that were further analyzed for their cellular phenotypes as described in RESULTS are marked by dots. (D) Growth of representative mutants was scored as described in C. Cells were scored ∼100 generations (five streaks) after the loss of the wild-type sequence. Cell were grown in liquid culture to OD₆₀₀ = 1.0 and serially diluted by 1:3. Equal volumes of the diluted cultures were spotted on the plate. The sequence and the telomere profile class for each mutant are indicated.

Figure 2.

Southern blots of one representative mutant from each class. Genomic DNA was prepared from cells after the indicated number of streaks, and then was digested with XhoI and probed with a wild-type telomeric repeat oligonucleotide. The sequences of positions 476–474 of TLC1 for each mutant are shown on the top. For pRS313 and 476gug, cells for last time point were ∼10 generations before senescence.

Figure 3.

Three mutants have high misincorporation rates. (A–C) Representative telomeric sequences cloned from three mutants. (D) Complete list of misincorporated sequences seen in three mutants. The TG-rich strand is shown. Correctly matched nucleotides that are synthesized from the mutant template are in lowercase. Misincorporated nucleotides are underlined and in bold.

Figure 4.

Template mutations lead to impaired chromosome separation and segregation. Strains bearing tlc1 template mutations contained Lac operator repeats integrated at the TRP1 locus near the centromere (yEHB5025-derived) or near the telomere (yEHB5026-derived). Template mutations were well-established at telomeres before cytological analysis (six passages, ∼120 generations). (A) Classes 1–7 show all spot-conformations that were observed during microscopic analysis in either cen- or telmarked strains. Sister chromatid separation and sister chromatid segregation were scored as indicated. (B) Distribution of marked chromosomes at representative time point, t = 100, for Wt cen; top2-4cen and top2-4tel; tlc1cen mutants (D) (E), and (SS); cdc13-1cen at 30°C and 23°C; cdc13-5cen; cdc13-1cen in combination with Δddc1, Δddc2, or both at 23°C and Δmad2 alone or in combination with tlc1cen (D).

Figure 5.

Analysis of chromosome dynamics. (A) Sister chromatid separation is delayed in the tlc1 template mutants, but there is no difference between centromere and telomere marked strains. (B) The number of chromosomes that fail to separate and segregate in the template mutants is high for all three classes, (D), (E), and (SS), but there is no difference between centromere- and telomere-marked strains.

Figure 6.

(A) Overall, budding is normal for tlc1(D), Δddc1, Δmec3, and tlc1(D) double mutants, but (B) there is an enrichment of large-budded cells in tlc1(D); tlc1(D)Δddc1; and tlc1(D)Δmec3. (C) The number of unsegregated chromosomes is as high in tlc1(D)Δddc1 and tlc1(D) Δmec3, as it is in tlc1-(D). Data for the 100-min time point is shown. (D) The number of cells with long spindles rapidly increases in DNA damage checkpoint mutants Δddc1 and Δmec3 but remains low in tlc1(D), tlc1(D)Δddc1, and tlc1(D) Δmec3 at the 80- and 100-min time points (E) The percentage of unsgegregated chromosomes remains high for all three template mutants, (D), (E), or (SS), when combined with Δddc2/Δsml1.