Multi-step coordination of telomerase recruitment in fission yeast through two coupled telomere-telomerase interfaces
- University of California, Irvine School of Medicine, United States
Figures
Figure 1 with 3 supplements
Fission yeast Tpz1 TEL-patch mutant leads to an EST phenotype.
(A) A sequence alignment of OB fold-domains between fission yeast Tpz1, human TPP1 and Oxytricha nova TEBP-β, in combination with a secondary structure prediction. Twelve conserved fission yeast …
Figure 1—figure supplement 1
Telomere length measurement of tpz1 mutant strains.
Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the indicated tpz1 mutant strains from successive re-streaks on agar …
Figure 1—figure supplement 2
Evaluation of Tpz1 expression levels in tpz1 mutant strains.
Western blot showing expression levels of Tpz1 protein in indicated tpz1 mutant strains.
Figure 1—figure supplement 3
The later generation tpz1-R81E/poz1Δ mutant strain forms circularized chromosomes.
(A) Schematic diagram of chromosome showing sub-telomeric regions. Greek numbers indicate the locations where the PCR primers are designed. (B) tpz1-R81E/poz1Δ mutant lost sub-telomeric I and II …
Figure 2 with 1 supplement
The Tpz1 TEL-patch mutant is defective in Tpz1-Trt1 interaction.
(A) Co-immunoprecepitation (Co-IP) assays evaluating bindings between Trt1 and Tpz1-E74R and Tpz1-R76E. Cdc2 was shown as the loading control. Input: 1/30 of input WCE (whole cell extract). (B) …
Figure 2—figure supplement 1
Trt1-Tpz1 interaction is intact in tpz1-K75E and tpz1-K75A strains.
Co-immunoprecepitation (Co-IP) assays evaluating the binding between full-length Trt1 and Tpz1 mutants K75E and K75A. Cdc2 was shown as the loading control. Input: 1/30 of input WCE (whole cell …
Figure 3
The Tpz1 TEL-patch mutant fails to localize telomerase to telomeres.
(A–C) Enrichment of Trt1 (A), Ccq1 (B) or Tpz1 (C) at telomeres is monitored by chromatin immunoprecipitation (ChIP) assay. Slot-blot was used to visualize telomere association of Trt1, Ccq1 or Tpz1 …
Figure 4
Fusing Trt1 to Tpz1 bypasses the requirement for functional Tpz1 TEL-patch.
(A) Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the trt1-tpz1 fusion strains with indicated WT or mutant versions of t…
Figure 5
Est1 binds to Ccq1 through two different binding sites.
(A) Ccq1-Est1 interaction is diminished but not completely disrupted in the ccq1-T93A, ccq1-F157A/K174E, and ccq1-I175R strains as evaluated by Co-IP assays. Cdc2 was shown as the loading control. …
Figure 6
Tpz1-Trt1 interaction is cell cycle-regulated.
(A) and (B) Co-IP assays evaluating the binding efficiency of Tpz1-Trt1 and Tpz1-Ccq1 interactions during cell cycle progression in ccq1+ (A) and ccq1-T93A (B) cells. In addition to the indication …
Figure 7 with 1 supplement
Model of cell cycle-regulated telomerase recruitment via an intermediate state with two coupled telomere-telomerase interactions.
A model showing telomerase recruitment in fission yeast through an intermediate state in which Trt1 and Est1 in the telomerase holoenzyme are collaboratively anchored to the telomere via Trt1-Tpz1 …
Figure 7—figure supplement 1
A speculative two-interface intermediate-state model for human telomerase recruitment.
A speculative two-interface intermediate-state model for human telomerase recruitment showing that the C-terminal domain of TIN2 is likely to be the Ccq1-functional equivalent in humans and …
Additional files
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Supplementary file 1
Strains used in the study and their genotypes.
- https://doi.org/10.7554/eLife.15470.015
