Telomere structures in SYn tlc1Δ survivors. Telomere Southern blotting assay was performed to examine telomere structure. The genomic DNA extracted from BY4742 (wild type) and SYn strains (labeled on top) was digested with XhoI, and subjected to Southern hybridization with a TG1-3 probe. (A) Telomerase-proficient strains (labeled on top), whose chromosome numbers are labeled at the bottom. Two independent clones of each strain were examined. (B-D) SYn tlc1Δ survivors generated on plates. Four (BY4742 tlc1Δ) and fifteen (SYn tlc1Δ) individual survivor clones (labeled on top of each panel) of each strain were examined. “+” at the bottom indicates Type I survivors. “Δ” marks the survivors which are non-canonical Type I or Type II.

Survivor formation in SY12 tlc1Δ strain. (A) Schematic representation of chromosome (and telomere) structures (not drawn to scale) in the SY12 strain (left panel) and the Type X survivor (right panel). The Roman numerals, native chromosomes; the Arabic numerals on the left, chromosome numbers of SY12; yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres; black circles, centromere; vertical arrows and numbers, positions and lengths of the terminal Xhol digestion fragments detected by the telomeric TG1-3 probe. Chromosome numbers are omitted in the Type X survivor (right panel). (B) Cell viability assay in liquid medium. The growth of SY12 (labeled in black) and SY12 tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (C) Telomeric Southern blotting assay of SY12 tlc1Δ survivors. Genomic DNAs prepared from SY12 tlc1Δ survivors assayed in (B) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (D) Telomere Southern blotting assay of SY12 tlc1Δ survivors obtained on solid medium. Genomic DNA from fifty independent SY12 tlc1Δ clones (labeled on top) was digested with XhoI and hybridized to a telomere-specific TG1–3 probe. Type II survivors: in orange; Type I survivors: in red; circular survivors: in blue; Type X survivors: in green; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12 strain are indicated on the left. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. The asterisks indicate the non-specific bands. Genomic DNA stained with Gelred was used as a relative loading control (LC). (E) Southern blotting results of an SY12 tlc1Δ Type II survivor at the 20th re-streaks after TLC1 reintroduction. LC: loading control. (F) Schematic of three circular chromosomes and fusion sequences in the SY12 tlc1Δ-C1 survivor. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres. (G) Telomere Southern blotting analysis of an SY12 tlc1Δ circular survivor at the 20th re-streak after TLC1 reintroduction. LC: loading control. (H) Telomere Southern blotting analysis of an SY12 tlc1Δ Type X survivor at the 20th re-streak after TLC1 reintroduction. The red arrows indicate the new band of about 4.3 kb emerged in Type X survivors. LC: loading control.

Figure 2—figure supplement 1. Characterization of SY12 strain. (A) Schematic representation of chromosome (and telomere) structures (not drawn to scale) in the SY12 strain. The Roman numerals, native chromosomes; the Arabic numerals on the left, chromosome numbers of SY12; yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres; black circles, centromere; vertical arrows and numbers, positions and lengths of the terminal Ndel digestion fragments detected by the telomeric TG1-3 probe. (B) Southern blotting analysis of telomere length in BY4742 and SY12 (labeled on top) cells. Genomic DNA prepared from two independent clones of BY4742 and SY12 strains was digested with NdeI, and then subjected to Southern blotting with a TG1-3 probe. The numbers in brackets indicate the telomere length of the corresponding chromosomes. The blot was then stripped and reprobed with a Y’ probe. The asterisk indicates the non-specific band.

Figure 2—figure supplement 2. Y’-elements have been eroded in SY12 tlc1Δ survivors. The blot in Figure 2D was then stripped and reprobed with a Y’ probe.

Figure 2—figure supplement 3. Borders of erosion of the SY12 tlc1Δ-C1 survivor are defined by PCR mapping. (A) Upper panel, a schematic diagram of the subtelomeric region of 0-60 kb proximal to I-L telomere is shown. Primer pairs (No. 1L to 17L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top; solid circles mean positive PCR products and open circles mean no PCR products with corresponding primer pairs. (B) Upper panel, a schematic diagram of the subtelomeric region of 0-50 kb proximal to X-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1R to 42R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (C) Upper panel, a schematic diagram of the subtelomeric region of 0-10 kb proximal to XIII-L telomeric TG1-3 sequence is shown. Primer pairs (No. 1L to 6L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (D) Upper panel, a schematic diagram of the subtelomeric region of 0-65 kb proximal to XI-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1R to 25R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (E) Upper panel, a schematic diagram of the subtelomeric region of 0-35 kb proximal to XVI-L telomeric TG1-3 sequence is shown. Primer pairs (No. 1L to 29L) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top. (F) Upper panel, a schematic diagram of the subtelomeric region of 0-60 kb proximal to XIV-R telomeric TG1-3 sequence is shown. Primer pairs (No. 1R to 21R) are aligned and indicated at their corresponding subtelomeric loci. Lower panel, the genotype is listed on the left, and primer pairs are listed on top.

Characterization of SY12YΔ, SY12XYΔ and SY12XYΔ+Υ strains. (A) Schematic of chromosome structures in the SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ strains. Yellow box, X-element; green box, Y’-element; tandem grey triangles, telomeres. Vertical arrows and numbers indicate the positions and sizes of the sites and length of Xhol and PaeI-digested terminal fragments. (B) PCR analyses of the engineered sites of the individual telomeres (labeled on left) in BY4742, SY12, SY12YΔ, SY12YΔ+1XΔ, SY12YΔ+2XΔ, SY12YΔ+3XΔ, SY12YΔ+4XΔ, SY12XYΔ and SY12XYΔ+Υ strains (labeled on top). Primer sequences for the PCR analyses are listed in supplementary file 1. RAP1 was an internal control. (C) Morphology of BY4742, SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ cells in the exponential growth phase (30°C in YPD). Shown are DIC images. Scale bar, 2 μm. (D) Growth analysis of the SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ strains. Several clones of the SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ strains were re-streaked on YPD plates 61 times at intervals of two days. Shown were the 3rd, 31st and 61st re-streaks. (E) Growth analysis of BY4742, SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ cells in liquid culture. Error bars represent standard deviation (s.d.), n = 3. (F) FACS analysis of DNA content of BY4742, SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ cells. (G) Dotting assays on YPD plates at low (24°C) and high (37°C) temperatures, or on YPD plates containing methyl methane sulfonate (MMS), camptothecin (CPT) or hydroxyurea (HU) at the indicated concentrations. The BY4742 mre11Δ haploid strain serves as a negative control.

Figure 3—figure supplement 1. Schematics of CRISPR–Cas9-mediated deletion of X- and Y’-elements on individual chromosomes in SY12 cells. The specific DNA sequences centromere-proximal to the subtelomeric region (site S1) were cleaved by the Cas9 nuclease with the guidance of gRNA1. Homologous recombination between the broken ends and the provided donors led to the deletion of X-and Y’-elements. Galactose induction of gRNA2 on pCas9 caused the cleavage at site S2 and the URA3 marker was counter-selected on 5’-FOA plates. Deletion of X- and Y’-elements were determined by PCR analysis with a primer located within the deletion region and another upstream of the region (indicated by purple arrows).

Telomere length and telomere silencing analyses of SY12YΔ, SY12ΧΥΔ and SY12XYΔ+Υ strains. (A) Southern blotting analysis of telomere length in SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ (labeled on top) cells. Genomic DNA prepared from three independent clones of SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ strains were digested with XhoI and PaeI, and then subjected to Southern blotting with a TG1-3 probe. The numbers in brackets indicate the telomere length of the corresponding chromosomes. (B) Expressions of MPH3 and HSP32 in BY4742, SY12, SY12YΔ, SY12XYΔ and SY12XYΔ+Υ cells were detected by qRT-PCR. The numbers above the schematic line (lower panels) indicate the distance to the corresponding subtelomeric elements or telomeres. The RNA levels of MPH3 and HSP32 were normalized by ACT1. The wild-type value is arbitrarily set to 1. Error bars represent standard deviation (s.d.), n = 3.

Survivor analysis of SY12YΔ tlc1Δ strain. (A) Cell viability assay in liquid medium. The growth of SY12YΔ (labeled in black) and SY12YΔ tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (B) Telomeric Southern blotting assay of SY12YΔ tlc1Δ survivors. Genomic DNAs prepared from SY12YΔ tlc1Δ survivors assayed in (A) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (C) Telomere Southern blotting analysis of SY12YΔ tlc1Δ survivors obtained on solid medium. Genomic DNAs of fifty independent survivors (labeled 1 to 50 on top) were digested with XhoI and hybridized by a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. Theoretical telomere restriction fragments of the SY12YΔ strain are indicated on left. LC: loading control. (D, F) Southern blotting results of an SY12YΔ tlc1Δ circular survivor (D) or an SY12YΔ tlc1Δ Type II survivor (F) at the 20th re-streaks after TLC1 reintroduction. LC: loading control. (E) Schematic of three circular chromosomes and fusion sequences in the SY12YΔ tlc1Δ-C1 survivor. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired, dashes are deleted. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres.

Figure 5—figure supplement 1. PCR mapping of the borders of erosion in SY12YΔ tlc1Δ-C1 cell. Red lines indicate the regions which have been deleted in the SY12YΔ strain.

Survivor analysis of SY12XYΔ tlc1Δ and SY12XYΔ+Y tlc1Δ strains. (A) Telomere Southern blotting analysis of SY12XYΔ+Y tlc1Δ survivors obtained on solid medium. fifty independent survivors (labeled 1 to 50 on top) were randomly picked, and their genomic DNAs were digested with XhoI and subjected to the Southern blotting assay with a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. The sizes of individual telomere restriction fragments of the SY12XYΔ+Y strain are indicated on the left. LC: loading control. (B) Telomeric Southern blotting assay of SY12XYΔ tlc1Δ survivors obtained on solid medium. fifty independent survivors (labeled 1 to 50 on top) were randomly picked, and their genomic DNAs were digested with XhoI and subjected to the Southern blotting assay with a TG1-3 probe. Type II survivors: in orange; circular survivors: in blue; atypical survivors: in black. The sizes of individual telomere restriction fragments of the SY12XYΔ+Y strain are indicated on the left. LC: loading control. (C) and (E) Schematic of three circular chromosomes and fusion sequences in the SY12XYΔ+Y tlc1Δ-C1 and SY12XYΔ tlc1Δ-C1 survivors, respectively. The sequence in blue indicates the sequences of X-R, XI-R or XIV-R, the sequence in red indicates the sequences of I-L, XIII-L or XVI-L. Bases in green are mis-paired, dashes are deleted. The numbers above or below the schematic line (chromosome) indicate the distance to the corresponding telomeres. (D) Southern blotting results of an SY12XYΔ+Y tlc1Δ circular survivor and an SY12 XYΔ+Y tlc1Δ Type II survivor at the 20th re-streaks after TLC1 reintroduction. LC: loading control. (F) Southern blotting result of an SY12XYΔ tlc1Δ Type II survivor at the 20th re-streaks after TLC1 reintroduction. LC: loading control.

Figure 6—figure supplement 1. SY12XYYΔ+Y tlc1Δ and SY12XYYΔ tlc1Δ strains form Type II survivors in liquid culture. (A) Cell viability assay in liquid medium: The growth of SY12XYYΔ+Y (labeled in black) and SY12XYYΔ+Y tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (B) Telomeric Southern blotting assay of SY12 XYYΔ+Y tlc1Δ survivors. Genomic DNAs prepared from survivors in (A) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe. (C) Cell viability assay in liquid medium: The growth of SY12XYYΔ (labeled in black) and SY12XYΔ tlc1Δ (three clones labeled in blue, purple and green respectively) strains were monitored every 24 hr for 12 days. (D) Telomeric Southern blotting assay of SY12 XYΔ tlc1Δ survivors. Genomic DNAs prepared from survivors in (C) were digested with XhoI and subjected to Southern blotting with a TG1-3 probe.

Figure 6—figure supplement 2. PCR mapping of the borders of erosion in SY12XYΔ+Υ tlc1Δ-C1 cell. Red lines indicate the regions which are absent in the SY12XYΔ+Υ strain.

Figure 6—figure supplement 3. PCR mapping of the borders of erosion in SY12XYΔ tlc1Δ-C1 cell. Red lines indicate the regions which have been deleted in the SY12XYΔ strain.

The synthetic lethality of YKU and TLC1 double deletion cannot be observed in “Circular survivors”. (A) Chromosomal DNA analysis of “circular survivors” SY12 tlc1Δ-C1, SY12YΔ tlc1Δ-C1, SY12XYΔ tlc1Δ-C1, SY12XYΔ+Y tlc1Δ-C1 by PFGE. The S. cerevisiae strain SY15 (with a single circular chromosome) was used as control. As marker, the size of three chromosomes in wild type S. pombe strain is indicated on right. (B) Serial dilution assay of yku70Δ tlc1Δ mutants. The isogenic strains (labeled on left) were spotted with five-fold dilutions on Ura- medium (left panel) to select for the presence of the pRS316-TLC1 plasmid, and on 5’-FOA medium (right panel) to select for the eviction of pRS316-TLC1 plasmid.