Cdc13 is predominant over Stn1 and Ten1 in preventing chromosome end fusions
- The State Key Laboratory of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, China
- School of Life Science and Technology, ShanghaiTech University, China
- Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
Successive passages of single-chromosome yeast strains SY14 and SY15 do not display growth change.
(A) Schematic of single chromosome structure in yeast strains SY14 and SY15. Single linear and circular chromosomes of the SY14 and SY15 strains are respectively aligned in the outer and inner …
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Figure 1—source data 1
Growth analysis of the SY14 and SY15 clones at the 3rd and 63rd re-streaks.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
The single chromosome in SY14 remains intact during the passages.
Fresh clones of the SY14 strain at different re-streaks (labeled on top) were inoculated into YPD and cultured overnight at 30°C, and pulsed-field gel electrophoresis (PFGE) analysis was performed. …
Figure 2 with 2 supplements
CST complex is not essential in single-circular-chromosome yeast strain SY15.
(A) Growth analysis of rap1Δ mutants. SY14 rap1Δ RAP1 and SY15 rap1Δ RAP1 cells were spotted with five-fold dilutions on Ura- medium (left panel) to select for the presence of pRS316-RAP1 plasmid, …
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Figure 2—source data 1
Growth analysis of the SY15 cdc13Δ, SY15 stn1Δ and SY15 ten1Δ cells.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Southern blotting to determine the insertion of TG255/CA255 sequence in SY15CA255 and SY15TG255 strains.
SY15CA255 and SY15TG255 strains (labeled at the top of each panel) were passaged on plates five times at intervals of two days. The genomic DNA was isolated from the 1st and 5th re-streaks (labeled …
Figure 2—figure supplement 2
The full sequences of the clones of SY15CA255 cdc13Δ (A) and SY15TG255 cdc13Δ (B) cells at different passages were shown as representatives.
The sequences comprise part of the KanMX marker (in green), a 255 bp telomeric tracts (in orange) and the upstream sequence of gene YJR154W (in grey). The XhoI restriction site is underlined. All …
Figure 3 with 4 supplements
Survivors of SY14 cdc13Δ mutant contain a circularized chromosome.
(A) Telomere Southern blotting analysis. 30 independent SY14 cdc13Δ colonies (labeled on top) were randomly picked, and their DNA was subjected to a telomere Southern blotting analysis to examine …
Figure 3—figure supplement 1
Borders of erosion and rTG Type of SY14 cdc13Δ survivors are defined by mapping and PCR amplification.
(A) Upper panel, schematic diagram of the subtelomeric region of 0–40 kb proximal to Chr XVI-L telomeric TG1-3 sequence is shown. Primer pairs (No. 1L to 30L) are aligned and indicated at their …
Figure 3—figure supplement 2
Fusion junctions in most SY14 cdc13Δ survivors contain TG sequences (rTG Type).
The sequences at the junction of 68 independent clones (labeled on left) of SY14 cdc13Δ survivors are determined: Figure 3—figure supplement 2, clone 1 to 38; Figure 3—figure supplement 3, clone …
Figure 3—figure supplement 3
Fusion junctions in most SY14 cdc13Δ survivors contain TG sequences (rTG Type).
Figure 3—figure supplement 4
Fusion junction sequences of non-TG Type survivors derived from SY14 cdc13Δ mutants.
The genotype and clone numbers are listed on right. Sequences at the fusion junctions of three independent clones are shown. According to the fusion sequences, they were be classified into three …
Figure 4 with 4 supplements
Chromosome fusion of SY14 cdc13Δ cells is nearly eliminated in the absence of Rad52.
(A) Schematic of rTG Type survivors in SY14 cdc13Δ. In SY14 cdc13Δ clone 30, the fusion region of TG1-3 sequence (in black) is in Chr XVI-L, and the (CA)17 region (in red) locates 43.9 kb away from …
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Figure 4—source data 1
Quantification of survivor generation rates of SY14 cdc13Δ, SY14 cdc13Δ rad52Δ, SY14 cdc13Δ yku70Δ and SY14 cdc13Δ pol32Δ cells.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig4-data1-v1.xlsx
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Figure 4—source data 2
NHEJ efficiency of SY14, SY14 lig4Δ and SY14 yku70Δ strains.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Survivors harboring circular chromosome maintain a stable genome.
Growth analysis of SY14 cdc13Δ (A) and SY14 tlc1Δ (B) survivors. A few clones of each strain were re-streaked at intervals of three days on YPD plates for 53 times.
Figure 4—figure supplement 2
Borders of erosion (A and B) and rTG Type (C) of SY14 cdc13Δ yku70Δ survivors are defined by mapping and PCR amplification.
Figure 4—figure supplement 3
Fusion junctions of rTG Type in SY14 cdc13Δ yku70Δ survivors.
The sequences at the junction of 29 independent clones (labeled on left) of SY14 cdc13Δ yku70Δ survivors are determined. In each clone, the length of the residual TG1-3 sequence proximal to junction …
Figure 4—figure supplement 4
NHEJ pathway is still functional in single-linear-chromosome yeast SY14.
(A) Map of the test plasmid substrate pRS316. The ampicillin marker, CEN/ARS cassette, URA3 marker and the multiple cloning site (MCS) containing EcoRI recognition sites are shown in green, black, …
Figure 5 with 6 supplements
Chromosome fusion frequency in either SY14 stn1Δ or SY14 ten1Δ cells is much lower than that in SY14 cdc13Δ cells.
(A) Quantification of survivor generation rates of SY14 cdc13Δ (1.14‰), SY14 stn1Δ (0.04‰) and SY14 ten1Δ (0.04‰) cells. Error bars represent standard deviation (s.d.), n = 3. (B) Percentage of rTG …
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Figure 5—source data 1
Quantification of survivor generation rates of SY14 cdc13Δ, SY14 stn1Δ, SY14 ten1Δ, SY14 cdc13Δ stn1Δ and SY14 cdc13Δ ten1Δ cells.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
PCR mapping of the borders of Chr XVI-L erosion in SY14 stn1Δ (A) and ten1Δ (B) survivors.
Figure 5—figure supplement 2
PCR mapping of the borders of Chr X-R erosion in SY14 stn1Δ (A) and ten1Δ (B) survivors.
Figure 5—figure supplement 3
Determination of rTG Type survivors by PCR (A and B) and fusion junction sequences of non-TG Type survivors (C) in SY14 stn1Δ and SY14 ten1Δ mutants.
Figure 5—figure supplement 4
PCR mapping of the borders of Chr XVI-L erosion (A) and Chr X-R erosion (B) in SY14 cdc13Δ stn1Δ and cdc13Δ ten1Δ survivors.
Figure 5—figure supplement 5
Determination of rTG Type survivors by PCR (A) in cdc13Δ stn1Δ and cdc13Δ ten1Δ survivors and fusion junction sequences of non-TG Type survivors (B) derived from SY14 cdc13Δ ten1Δ mutants.
Figure 5—figure supplement 6
SY14CA255 cstΔ survived cells utilize CA255 sequence for chromosomal circularization.
(A) Schematic representation of SY14CA255 strain. A 255 bp telomeric sequence (named CA255, tandem orange triangles) is inserted between the PGU1 and YJR154W genes. These two genes are located at …
Figure 6 with 4 supplements
Telomerase inactivation in SY14 cells results in senescence and survivor formation.
(A) Senescence assay in liquid medium. The growth (OD600) of SY14 (green), SY14 tlc1Δ (three clones in pink, red and orange), SY15 (black) and SY15 tlc1Δ (blue) strains were monitored every 24 hr …
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Figure 6—source data 1
Senescence assay of SY14 tlc1Δ and SY15 tlc1Δ cells.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
PCR mapping of the borders of Chr XVI-L erosion (A) and Chr X-R erosion (B) in SY14 tlc1Δ survivors.
Figure 6—figure supplement 2
Determination of rTG Type survivors by PCR (A) and fusion junction sequences of non-TG Type survivors (B) of SY14 tlc1Δ mutants.
Figure 6—figure supplement 3
Fusion junctions of rTG Type in SY14 tlc1Δ survivors.
The sequences at the junction of 42 independent clones (labeled on left) of SY14 tlc1Δ survivors are determined. In each clone, the length of the residual TG1-3 sequence proximal to junction point …
Figure 6—figure supplement 4
Telomerase null cells may experience inter- and/or intra-chromosome fusions to survive with the decrease of chromosome numbers in budding yeast.
(A) Telomere Southern blotting analysis. The genomic DNAs from the strains (labeled on top) with different numbers of chromosomes (labeled at bottom) were digested by XhoI and subjected to Southern …
Figure 7 with 5 supplements
Survivors in SY14 tlc1Δ have a circular chromosome.
(A) Senescence assay in liquid medium. The growth (OD600) of SY14 (black), SY14 tlc1Δ (red, orange) and SY14 tlc1Δ rad52Δ (green, blue) strains were monitored every 24 hr for 15 days. (B) Telomere …
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Figure 7—source data 1
Senescence assay of SY14 tlc1Δ and SY14 tlc1Δ rad52Δ cells.
- https://cdn.elifesciences.org/articles/53144/elife-53144-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
PCR mapping of the borders of Chr XVI-L erosion in SY14 tlc1Δ rad52Δ survivors.
Figure 7—figure supplement 2
PCR mapping of the borders of Chr X-R erosion in SY14 tlc1Δ rad52Δ survivors.
Figure 7—figure supplement 3
Fusion junction sequences of non-TG Type survivors derived from SY14 tlc1Δ rad52Δ mutants.
Figure 7—figure supplement 4
Borders of erosion (A and B) and rTG Type (C) of SY14 tlc1Δ yku70Δ survivors are defined by mapping and PCR amplification.
Figure 7—figure supplement 5
Chromosomal circularization in SY14 tlc1Δ cells is independent of Pol32.
(A-C) Borders of erosion (A and B) and rTG Type (C) of SY14 tlc1Δ pol32Δ survivors are defined by mapping and PCR amplification.
Additional files
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Supplementary file 1
Mapping primers used in this study.
- https://cdn.elifesciences.org/articles/53144/elife-53144-supp1-v1.docx
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Supplementary file 2
Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/53144/elife-53144-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53144/elife-53144-transrepform-v1.docx
