Fig 1.
Measurement of spontaneous GCR rates in fission yeast.
(A) Schematic structure of the ura4-TK strain for the GCR assay. ura4+ and TK, which confer sensitivity to 5-fluoroorotic acid (5-FOA) and 5-fluoro-2'-deoxyuridine (FUdR), respectively, were inserted in the right arm of chromosome I. sec16+ is the most distal essential gene on the right arm. (B) GCR rates, which are median rates for the accumulation of 5-FOA/FUdR-resistant progeny, are plotted on a log scale for each strain. In all the figures showing GCR rates in this report, error bars represent the >95% confidence intervals. (C) Numbers of different types of GCRs identified in 5-FOA/FUdR-resistant clones derived from wild-type strain. (D) Representative breakpoint junction sequences in GCR survivors derived from wild-type cells. In the case of a deletion type survivor (upper panel), telomeric repeats are indicated by lowercase letters in red. The number in brackets indicates the nucleotide coordinate of the chromosome I at which the telomere repeats were added. In the case of a translocation type survivor (lower panel), the upper and lower sequences are the original chromosomal DNA sequences proximal to the translocation, and the underlined nucleotides indicate the fused sequence. The red letters indicate identity. The reference sequence was obtained from the Pombase website (http://www.pombase.org/). See S1B Fig and S2 Table for the detailed sequential changes. (E) Distribution of GCR junctions in the breakpoint region. Each arrowhead represents the location of a junction in the 5-FOA/FUdR-resistant clones. Closed and open arrowheads indicate deletion and translocation types, respectively.
Fig 2.
Regulation of GCRs by individual telomere binding proteins.
(A) Schematic representation of the S. pombe shelterin complex. (B) GCR rates of telomere-binding protein mutants. (C) Numbers of different types of GCRs identified in 5-FOA/FUdR-resistant clones derived from strains with the indicated genetic backgrounds. (D) Distribution of GCR (exclusively deletion types) junctions in the breakpoint region. Each arrowhead represents the location of a junction in the 5-FOA/FUdR-resistant clones. (E) GCR rates of poz1Δ and tpz1-I501A/R505E mutants in wild-type, taz1Δ, and rap1Δ backgrounds. Asterisks represent a statistically significant difference (P < 0.05) from the wild-type strain, unless otherwise noted. NS: non-significant (P > 0.05). P-values were determined by the two-tailed Mann–Whitney test.
Fig 3.
Effects of telomere end-to-end fusion and telomere tethering to the nuclear envelope on GCR formation.
(A) GCR rates of wild-type, taz1Δ, and rap1Δ strains in the presence or absence of lig4+. (B) GCR rates of strains with INM protein mutants. GFP-Bqt4ΔN or Rap1-GFP-Bqt4ΔN fusion protein was expressed from the bqt4 promoter integrated into genomic DNA as described previously [23]. (C) GCR rates of wild-type, taz1Δ, and rap1Δ strains in the presence or absence of bqt4+. Asterisks represent significant differences (P < 0.05) from the wild-type strain unless otherwise noted. P-values were obtained by the two-tailed Mann–Whitney test.
Fig 4.
Telomerase activity is essential for frequent GCRs in taz1Δ and rap1Δ strains.
(A) GCR rates of wild-type, taz1Δ, rap1Δ, and rad2Δ strains in the presence or absence of pof8+. (B) GCR rates of trt1Δ strains in the presence or absence of a pREP1-trt1+ plasmid (Trt1 plasmid). The GCR assay was performed in YES media, in which the nmt1 promoter in the plasmid has a moderate activity [69]. Asterisks and NS represent significant differences (P < 0.05 and P>0.05) from the wild type strain, respectively. P-values were obtained by the two-tailed Mann–Whitney test.
Fig 5.
The Poz1-binding domain and BRCT domain of Rap1 redundantly suppress GCRs.
(A) Schematic of Rap1 truncation mutants. (B) GCR rates of the Rap1 mutants shown in (A). Rap1 proteins were expressed from a single copy of cognate alleles integrated downstream of the native rap1 promoter. FL, full length. Asterisks represent significant differences (P < 0.05) from the wild type strain. P-values were obtained by the two-tailed Mann–Whitney test. (C) Numbers of different types of GCRs identified in 5-FOA/FUdR-resistant clones derived from wild-type strain.
Fig 6.
Taz1 and Rap1 are involved in the repair of I-SceI-induced DSBs.
(A) Schematic of our site-specific DSB induction system. A gene that encodes endonuclease I-SceI was placed under a modified CaMV 35S promoter that can be repressed by the Tet repressor (TetR) [67]. I-SceIcs was inserted at the same site as the marker gene cassette in the ura4-TK strain. A DSB at I-SceIcs can be induced by TetR inhibition via ahTET treatment. (B) Efficiency of DSB induction in I-SceIcs. At the indicated time points after addition of ahTET, DNA breaks were monitored by qPCR with a primer set flanking I-SceIcs. (C) Frequencies of colony-forming cells on non-selective agar plates with or without ahTET treatment for 2 hours. Asterisks represent significant differences (P < 0.05) from the wild-type strain. P-values were obtained by the two-tailed Student’s t-test. We found that I-SceI digestion proceeded for additional two hours ahTET washout (S6A Fig), likely due to residual I-SceI activity remaining after the expression shut-down. Therefore, the digestion efficiencies were likely even greater than the 40~50% indicated in (B). (D) Current model of how Rap1 functions in DSB repair and GCR suppression. See text for details. (E) Survival rates determined by counting numbers of colonies formed on ahTET-containing and non-selective agar plates. (F) Locations of the GCR junctions in the survivors isolated in (E). All survivors were examined for GCR structures as in Fig 1C and 1E. All clones had chromosomal terminal deletions, and none of them displayed translocations. We could not identify the GCR type and location of the junction in one out of the ten examined survivors in both the WT and taz1Δ strains. Each triangle (arrowhead) represents the location of the GCR junctions. The inset shows the sequences around I-SceIcs. The cleavage pattern of I-SceI is shown with a line. Regarding (B), (C), and (E), mean values ± SEM are shown. N = 3.