Telomere fusions caused by mutating the terminal region of telomeric DNA

Abstract

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.

Figure 1

TER1 template mutations used in this study. Shown is the 25-nt sequence of the K. lactis telomeric repeat (the strand and permutation that is synthesized by telomerase). The overlined nucleotides represent the Rap1 binding site. The BsF mutation was generated from a partial filling in the overhang of the BsiWI site present in the ter1-Bsi mutant (13). All other alleles have names that include the restriction enzyme site(s) produced by the mutation. The G to A change of ter1-AccSna produces an AccI site whereas the G to T change produces a SnaBI site. The latter change, by itself, constitutes the ter1-Sna allele.

Figure 2

Telomeric changes in ter1-BsF cells. (A) Southern blot of ter1-BsF mutant followed over 14 serial streaks after its creation (as numbered above lanes). EcoRI-digested genomic DNA was first hybridized with a probe specific to BsF mutant repeats (Left). After stripping, this filter was rehybridized with a probe specific to wild-type telomeric repeats (Center). This detects telomeres that have yet to have BsF repeats added onto them as well as telomeres elongated by the BsF mutant telomerase. Another filter with DNA from streaks 5, 8, 11, and 14 from the same clone probed with an oligonucleotide that binds equally to both wild-type and BsF repeats is shown (Right). The telomeric pattern of wild-type cells can be compared from B. (B) Southern blot showing telomeres of a single clonal lineage of ter1-BsF cells followed over 40 serial streaks on rich media. Genomic K. lactis DNA was digested with EcoRI and probed with a wild-type K. lactis telomeric oligonucleotide. DNA from the wild-type parental control is also shown. (C) Bal31 exonuclease time course done with DNA from ter1-BsF and ter1-AccSna mutants after formation of sharp bands carrying telomeric repeats. Numbers above lanes indicate length of Bal31 treatment in minutes. After exonuclease treatment, samples were digested with EcoRI before electrophoresis and Southern blotting. Hybridization was with a telomeric probe. Rate of disappearance of telomeric signal paralleled disappearance of total DNA in both mutants as seen on ethidium bromide straining of the gel, whereas disappearance of telomeric signal in wild type preceded complete digestion of total DNA (not shown).

Figure 3

Telomeric changes in four TER1 template mutations that disrupt the Rap1 binding site in telomeric repeats. (Left) Southern blots of the parental wild-type strain and ter1 mutants at one streak after their isolation. (Right) Southern blots of the same mutants after 20 streaks (except ter1-AccSna mutants, which are shown after 32 streaks). Two independent isolates of each mutant are shown. Each isolate is shown digested with EcoRI alone (−) or with EcoRI plus an enzyme that cleaves off the mutant repeats (+) (BglII for 8LR:BsrBgl and 4R:Bgl mutants, BsrGI for 4LR:Bsr mutant, and AccI for AccSna mutant). The double digest of the wild-type strain was EcoRI plus AccI. Note that AccI cleaves the largest EcoRI telomeric fragment at a subtelomeric position. The left clone of ter1–4R:Bgl is shown darkly exposed to show the signal present at limit mobility (“L”) and in the well. Probe used was a telomeric oligonucleotide, Klac1–25. Size markers are shown between panels. Below is a diagram showing the basic structure of telomeres in mutants with short, long, or fused telomeres. White and gray boxes indicate wild-type and mutant telomeric repeats, respectively. Arrows indicate the direction of the telomeric end or, on the fused telomere, the direction of the telomeric ends before fusion. “E” indicates the EcoRI site nearest the telomere, the actual position of which varies between different telomeres.

Figure 4

Formation of telomere fusions after introduction of a linear plasmid. (A) A Southern blot hybridized with a telomeric probe of EcoRI-digested DNA isolated from the first streak after cells were transformed with the K. lactis linear plasmid pHisLin1. The lower part of the gel is shown more darkly exposed. Both telomeres of the linear plasmid run at the same position (≈0.5 kb) in wild-type TER1 cells (lanes 7 and 8). Six independent small colony transformants of pHisLin1 into ter1-AccSna are shown in lanes 1–6. Arrow indicates the position of short, unfused telomeres visible in some of the ter1-AccSna transformants. (B) Southern blots of ApaLI + NgoMIV-digested DNA from some of the same clones after five serial restreaks hybridized with probes unique to either the left or the right subtelomeric region of the transformed linear vector. Numbers above lanes match clone designations from A. Diagram above shows the inferred map of a typical telomere fusion. White and gray boxes indicate wild-type and mutant telomeric repeats, respectively. Arrows indicate the direction of the telomeric ends before fusion. Hatched boxes indicate regions used as probes and black bar indicates 200 bp. N, E, and A indicate NgoMIV, EcoRI, and ApaLI restriction sites.