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. 2012 Mar 2;148(5):922-32.
doi: 10.1016/j.cell.2012.01.033. Epub 2012 Feb 23.

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

Jennifer S Pfingsten  1 , Karen J Goodrich, Cornelius Taabazuing, Faissal Ouenzar, Pascal Chartrand, Thomas R Cech
Affiliations

Mutually exclusive binding of telomerase RNA and DNA by Ku alters telomerase recruitment model

Jennifer S Pfingsten et al. Cell. .

Abstract

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:

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Figures

Figure 1
Figure 1
Truncations within the Ring of Ku80 Impair NHEJ and Silencing (Telomere Position Effect) (A) Internal deletions within the loop that slides over the dsDNA were designed on the basis of the crystal structure of human Ku (Walker et al., 2001). (B) Using a strain with an engineered HO endonuclease cut-site, the Ku mutants, along with the WT strain and Δku80, were streaked onto plates containing either glucose or galactose. Galactose induces the production of HO endonuclease, triggering dsDNA break repair in cells containing functional Ku. The cells were plated at 100 generations. (C) Western blot of the mutant yeast protein content after 20 generations. The loading control is DSK2, an endogenous protein. The Ku80 mutant to WT protein ratios (n = 4) were: Ku80Δ4 0.9 ± 0.1; Ku80Δ12 0.7 ± 0.3; Ku80Δ20 0.8 ± 0.3; Ku80Δ28 1.2 ± 0.6; Ku80Δ36 1.0 ± 0.3; Ku80Δ40 1.3 ± 0.4. (D) Silencing assay for the URA3 gene at 20 generations. This strain is described in Experimental. See also Figure S1.
Figure 2
Figure 2
Mutations within Ku's DNA-binding Loop Reduce Telomere Length (A) Southern blot of Xho1-linearized yeast genomic DNA shows the length of Y' telomeres (heterogeneous distribution) and non-Y' telomeres (discrete bands at 2 kbp and above) over the course of generations 20, 100, and 200. The * symbol denotes the loading control, which is a restriction fragment of Chromosome IV. (B) Measurement of average telomere length. The triangle represents 20, 100, and 200 generations. See also Figure S2.
Figure 3
Figure 3
Ku80Δ28 Protein Loses Affinity for Both dsDNA and TLC1-KBS RNA (A) Binding of 22-bp dsDNA with a 14-nt telomeric 3' overhang by purified WT Ku and Ku80Δ28 proteins assayed by EMSA. (Throughout this paper, Ku80Δ28 protein refers to a heterodimer of Ku70 and Ku80Δ28.) Lanes 1 – 6 contain the following amounts of active WT Ku protein: 0; 0.02 nM; 0.2 nM; 1.7 nM; 17.2 nM; 172 nM. Lanes 7 – 12 contain active Ku80Δ28 protein in the same amounts as in lanes 1 – 6. (B) Binding of TLC1-KBS RNA to WT Ku and Ku80Δ28 assayed via EMSA. The amounts of active protein are the same as in (A). (C) Graphical representation of the DNA binding seen in (A). The active Kd for WT Ku is 0.08 nM. The active Kd for Ku80Δ28 is 0.78 nM. (D) Graphical representation of (B). The fitted data yielded active Kd = 4.9 nM for WT Ku and active Kd = 87.1 nM for Ku80Δ28 binding to TLC1-KBS RNA. See also Figure S3.
Figure 4
Figure 4
Ku80Δ28 Loses Association with TLC1 RNA in vivo and the RNA Accumulates in the Cytoplasm (A) TLC1 RNA immunoprecipitation with Myc-tagged Ku proteins analyzed by real-time RT-PCR. Cells were subjected to formaldehyde crosslinking to preserve RNA-protein interactions prior to immunoprecipitation on anti-Myc beads. The highest levels of pull-down (around 15-fold enrichment) corresponded to 2% of the input TLC1 RNA. ACT1 mRNA, which is not known to associate with Ku, served as a control for nonspecific binding. Bars indicate average of five biologic replicates performed on four different weeks, and error bars give SEM. (B) Localization of endogenous TLC1 RNA in WT, Δku80 and ku80Δ28 strains was detected using fluorescent in situ hybridization. DAPI: DNA staining. Scale bar: 1 μm. (C) Quantification of TLC1 RNA distribution in WT, Δku80 and ku80Δ28 strains. For each strain, a total of 300 cells were randomly scored, in three independent experiments. See also Figure S4.
Figure 5
Figure 5
Mutually Exclusive Binding of RNA and DNA to WT Ku (A) A mixing experiment shows the complexes that Ku forms with dsDNA containing different 3' overhangs and with TLC1-KBS RNA. Each arrow denotes the position in the gel for a particular complex. (B) TLC1-KBS RNA and DNA compete against one another to bind Ku. Increasing amounts of TLC1-KBS RNA were added to samples containing Ku and radiolabeled dsDNA with different 3' overhangs. The fraction bound of each sample was calculated and fitted to a single binding site competition formula to calculate the Ki of the RNA. (C) 100 nM of TLC1-KBS RNA and 3xmutTLC1-KBS RNA were added in equilibrium binding experiments to ascertain their effects on Ku's affinity for radiolabeled dsDNA with telomere-like 3'overhang. The fraction bound of the DNA was calculated and fitted to the Langmuir isotherm. See also Figures S5 and S6.
Figure 6
Figure 6
Models of Ku's Role in Telomerase Recruitment (A) Published model shows Ku binding simultaneously to TLC1 RNA and the dsDNA while recruiting telomerase to the chromosome end, which is not possible based on our work. (B) Based on the action of Ku during NHEJ, telomere-bound Ku might bind to a telomerase-bound Ku to recruit telomerase to telomeres. However, the Ku70 separation-of-function mutants described by Ribes-Zamora et al. (2007) and data presented herein cause us to discount this model. (C) In the new model, Ku recruitment of telomerase begins with its key role in nuclear import and retention. When telomerase-bound Ku encounters telomeric DNA, Ku may be handed off from TLC1 to the DNA (blue line). This hand-off may be necessary to prevent telomerase from being sequestered in telomeric heterochromatin by Ku-Sir4 binding. The Est1-Cdc13 protein-protein interaction then secures telomerase to the telomere. Other reported interactions include Est1 and Sir4 binding to the nuclear envelope protein Mps3 (black dashes) and Ku and Cdc13 binding to Sir4 (red dashes). See also Figure S7.
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