Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends

Abstract

Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends. At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Here, we present evidence of a new role for Ku that impacts both telomere-length maintenance and DNA repair in Saccharomyces cerevisiae. We show that Ku binds TLC1, the RNA component of telomerase. We also describe a novel separation-of-function allele of Ku that is specifically defective in TLC1 binding. In this mutant, telomeres are short and the kinetics of telomere addition are slow, but other Ku-dependent activities, such as chromosome end protection and NHEJ, are unaffected. At low frequency, yeast will use telomerase to heal DNA damage by capping the broken chromosome with telomeric DNA sequences. We show that when Ku's ability to bind TLC1 is disrupted, DNA repair via telomere healing is reduced 10- to 100-fold, and the spectrum of sequences that can acquire a telomere changes. Thus, the interaction between Ku and TLC1 RNA enables telomerase to act at both broken and normal chromosome ends.

Figure 1.

Ku binds the TLC1 RNA stem-loop in vitro. (A) Purification of Ku from yeast. A Coomassie-stained protein gel is shown, containing a protein molecular weight ladder and 1 μg of the pooled eluate from the second round of affinity purification. (B) Secondary structure of the TLC1 RNA stem-loop. Nucleotides 288-335 of the 1.3-kb TLC1 RNA are shown. Circles indicate the residues that were changed (U301A, U307G, U324G) in the mutant RNA substrate used in C. Figure adapted from Peterson et al. (2001), and reproduced with permission from Nature Publishing Group. (C) Gel-shift analysis of Ku's RNA-binding activity. Reactions contained 12 pM wild-type stem-loop RNA (lanes 1-5) or 13 pM mutant RNA (lanes 6-10). Ku was present at 0 nM (lanes 1,6), 0.2 nM (lanes 2,7), 1 nM (lanes 3,8), 5 nM (lanes 4,9), and 25 nM (lanes 5,10).

Figure 2.

Isolation of a new yku80 allele that suppresses the effect of TLC1 overexpression on telomeric silencing. (A) Schematic of the reporters and phenotypes of telomeric silencing. ADE2 and URA3 are located near the telomeres (jagged lines) of chromosomes V and VII, respectively. (B) Observed telomeric silencing of UCC6058 strains containing the plasmids indicated below. Tenfold serial dilutions of each strain were plated onto YC-trp-leu + GAL medium to evaluate colony color (gray = pink) and number, and onto YC-trp-leu-ura + GAL medium to evaluate growth in the absence of uracil. (Row 1) pRS315 + pTCG. (Row 2) pRS315-YKU80 + pTCG. (Row 3) pRS315-YKU80 + pTCG-3X stem. (Row 4) pRS315-yku80-135i + pTCG-3X stem.

Figure 3.

Mutant Ku protein binds DNA but not the TLC1 stem-loop RNA in vitro. (A) Gel-shift analysis of RNA-binding reactions. A total of 16 pM wild-type TLC1 stem-loop RNA was used as a substrate. Ku was present at 0 nM (lanes 1,5), 0.5 nM (lanes 2,6), 8 nM (lanes 3,7), and 40 nM (lanes 4,8). (B) Gel-shift analysis of DNA-binding reactions. A total of 190 pM ADE2 DNA was used as a substrate. Two shifted species are seen, probably reflecting Ku binding to one or both ends of the DNA fragment. Ku concentrations were the same as in A. (C) Structure of human Ku bound to DNA. Ku70 is in yellow, Ku80 is in blue, the DNA is in white, and the residues that correspond to the site of the 15-bp insertion are in red. Figure prepared using Swiss PBD Viewer and the coordinates from accession number 1JEY in the Protein Data Bank (Walker et al. 2001).

Figure 4.

DNA end protection and NHEJ are not compromised in yku80-135i strains. (A) Analysis of telomere end structure. Two independent isolates of UCC6058 strains containing pRS315-YKU80, pRS315-yku80-135i, or pRS315 were analyzed. Genomic DNA was digested with XhoI and resolved by agarose gel electrophoresis, along with a DNA molecular weight ladder. The gel was hybridized with a radiolabeled C_1-3A probe first under nondenaturing conditions and then under denaturing conditions. The terminal restriction fragments of chromosomes with subtelomeric Y′ elements are 1.0-1.3 kb in size (depending on strain background); chromosomes without Y′ elements give rise to terminal restriction fragments of diverse sizes. (B) Analysis of temperature sensitivity. Growth at 37°C of the strains in A was compared. (C) Transformation efficiencies of UCC5913, UCC3744, and UCC3745. Values represent the number of transformants recovered with linearized pBTM116 relative to the number recovered with supercoiled pBTM116. Experiments were done in triplicate.

Figure 5.

Telomere length maintenance and de novo telomere addition are affected when Ku cannot interact with TLC1 RNA. (A) Southern blot analysis of telomere length in UCC5114, UCC5116, UCC5120, and UCC5118-2. The positions of relevant DNA size markers are indicated. (B) Schematic of the de novo telomere addition assay. The left arm of chromosome VII contains a short telomeric seed sequence (thin jagged line) and a recognition site for the HO endonuclease (gray box) next to an ADE2 marker. Upon induction of HO, the chromosome is cleaved to expose the telomeric seed sequence. Telomerase then adds new telomeric repeats onto it (thick jagged line). (C) Southern blot analysis of de novo telomere addition in UCC5913, UCC6073, UCC3744, and UCC3746. Blots were probed with an ADE2 fragment that also hybridizes to a 1.6-kb fragment from the genomic ade2-101 locus (labeled “int”), which serves as an internal loading control. The product of HO cleavage (labeled “cut”), and the smear of products reflecting new telomere addition (marked with a bar) are also indicated.

Figure 6.

The Ku-TLC1 interaction affects telomere healing events. (A) Schematic of the relevant markers on chromosome VL. URA3 replaced the endogenous HXT13 gene, located -7.5 kb from CAN1 and -22 kb from the end of the chromosome. (B) Southern blot analysis of representative GCR events. Three telomere healing events (lanes 1,3,4) and one translocation event (lane 2) are shown. (C) The NPR2 hotspot. Nucleotides 451-473 are shown. Positions of telomere addition, and the number of independent events recovered, are indicated for wild-type strains (downward arrows) and all mutant strains (upward arrows). (D) Chromosome V sequences at the junctions of unique telomere addition events from wild-type and all mutant strains. Sequences run 5′-3′; telomeres were added proximal to the last residue indicated. (E) Histogram quantifying the number of TG residues at the junctions of the telomere addition events in D. Contiguous T₁G_1-3 residues were counted; one intervening non-T₁G_1-3 residue was allowed to be skipped. The skipped residue was not included in the count, and only one skip was made per sequence.