Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Abstract

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.

Fig. 1.

K. lactis telomerase RNA and mutations used in this study. A map of the telomerase RNA gene (TER1) shows the locations of the 30-bp template (open rectangle) and the ter1-Δ1051-1242 mutation (shaded rectangle). The sequence of telomeric DNA complementary to telomerase RNA is shown below for the wild type (WT) and ter1 mutants. The first and last bases of the 30-bp template region are indicated by black dots below the WT sequence. The TTTGA terminal repeats of the template (underlined) are necessary for accurate telomerase alignment and binding during translocation events. Single-nucleotide mutations are underlined; deletions are represented by underlined hyphens, and insertions are indicated by asterisks. The phenotypically silent 7C(Bcl) and 20C(ApaL) mutations create BclI and ApaLI restriction sites, respectively. The ter1-24T(SnaB) strain carries a single base change creating a SnaBI restriction site within the telomeric Rap1-binding site that also affects the translocation step of telomerase (65). The ter1-Dup21-25 strain has a permutated template shifted 5 bp that is predicted to make wild-type telomeric repeats. The new 5-bp terminal repeats of this template are indicated by dashed underlines. The equivalent single copy of this sequence is also indicated in the WT and other mutants. The ter1-28C(Taq) mutant contains a base change in the 5′-terminal direct repeat of the TER1 template interfering with telomerase translocation and leading to the synthesis of 31-bp repeats (64).

Fig. 2.

Deletion of RAD52 exacerbates growth defects in some ter1 mutants with short telomeres. (A) Colony growth of a wild-type control strain (TER1 RAD52) and the ter1-Δ1051-1242 mutant, shown as examples of normal colony morphology and abnormal rough-colony morphology, respectively. (B) Photographs of clonal passaging of ter1-Δ, ter1-ΔR-Bcl, and ter1-Δ1051-1242 single mutants and double mutants with rad52-Δ on solid rich YPD medium.

Fig. 3.

The poorest colony morphology correlates with the shortest telomere length. Shown is a Southern blot of genomic DNA, hybridized to a telomeric probe, from the parental wild-type (WT) strain and two independent clones of each mutant (indicated by horizontal lines above lanes). DNA was digested with BsrBI and was resolved on a 3.2% agarose gel.

Fig. 4.

Smooth-colony derivatives of the ter1-Bgl(fill) mutant have telomeres lengthened by recombination. (A) Genomic DNA was isolated from three independent clones of the ter1-Bgl(fill) mutant with smooth-colony morphology and a control ter1-Bgl(fill) mutant with the typical slightly rough colony morphology. DNA was digested with EcoRI and also with ClaI (indicated by plus signs above lanes) prior to Southern blotting and hybridization to a telomeric probe. An inset at the bottom right depicts further resolution of bands released by EcoRI and ClaI digestion in the three clones by use of a 6% polyacrylamide gel. (B) Diagram showing a segment of telomeric DNA composed of wild-type (WT) and mutant repeats (shaded and open rectangles, respectively). Cleavage of Bgl(fill) mutant repeats by ClaI (indicated by arrows) excises fragments composed of WT repeats with terminal halves of mutant repeats as well as 29-bp mutant repeat fragments. The latter are not retained on the hybridization membrane.

Fig. 5.

Smooth-colony derivatives of the ter1-ΔR-Bcl mutant exhibit lengthened telomeres, some with amplification of small blocks of wild-type (WT) repeats. Shown is a Southern blot of genomic DNA, hybridized to a telomeric probe, from five independent ter1-ΔR-Bcl clones, initially identified as having smoother than normal colonies. DNA was digested with EcoRI and then additionally with BclI (indicated by plus signs above lanes).

Fig. 6.

Smooth-colony derivatives of the ter1-ΔR-Bcl mutant undergo gradual telomere shortening and return to a rough-colony phenotype with continued passaging. Four clones of ter1-ΔR-Bcl with elongated telomeres were serially passaged over eight streaks. DNA was cut with EcoRI prior to Southern blotting and hybridization to a telomeric probe. Graphs above Southern blots represent colony morphologies. A score of 1 represents the poorest growth with rough colonies, while a score of 4 defines a wild-type-like colony phenotype with completely smooth colonies (37). Arrows in blots represent instances where a sizable increase in the length of one or more telomeres has occurred in a cell population but apparently not in the cell from that streak that became the source of cells for the next streak. Data for the wild-type (WT) parental strain are shown in the panel with clone 4.

Fig. 7.

Smooth-colony derivatives of the ter1-Δ1051-1242 mutant have telomeres lengthened by recombination. (A) Southern blots of two clones of the ter1-Δ1051-1242 mutant with typical rough colonies. Genomic DNA was digested with EcoRI and then also with BclI (indicated by plus signs above the lanes) prior to hybridization with a telomeric oligonucleotide that hybridizes to wild-type (WT) (and mutant) telomeric repeats. The first two streaks of each clone are shown. (B) The filter shown in panel A was rehybridized with the Klac Bcl probe specific to Bcl telomeric repeats. (C) Three better-growing clones with smooth-colony morphology were examined using the same digestions and probe as in panel A. The inset at the bottom shows further resolution of the bands released by digestion with EcoRI and BclI in three clones by use of a 3.2% agarose gel.

Fig. 8.

Mutant cells undergoing RTE passaged in liquid medium. A wild-type (WT) control and two clones each of the ter1-Δ, ter1-ΔR-Bcl, and ter1-Bgl(fill) strains were passaged nonclonally for 20 serial dilutions in liquid YPD medium. Shown are Southern blots of genomic DNA isolated from these strains at different passages (indicated by numbers above lanes), digested with EcoRI, and subsequently hybridized to a telomeric probe.