The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini

Abstract

The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80(tel) alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80(tel) alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

FIG. 1.

yku80^tel mutants are proficient for repair of DSBs. (A) In vivo plasmid repair assay. A yku80-Δ rad52-Δ haploid strain bearing plasmid-borne yku80^tel mutant alleles was transformed with a mixture containing EcoRI, linearized pRS413 (CEN HIS3), and supercoiled pRS415 (CEN LEU2). Mutant alleles were tested in parallel with wild-type YKU80 and vector controls. The repair efficiency is the Ura⁺/Leu⁺ transformant ratio normalized to that of YKU80. The results are the averages of the results of 2 to 4 transformations per strain from three separate experiments. Each error bar represents one standard deviation. (B) Continuous HO expression assay. Plasmids bearing yku80^tel mutant alleles were transformed into YVL2073 (yku80-Δ::kan^r hml-Δ::ADE1 hmr-Δ::ADE1 ADE3::GAL:HO) (Table 1). Tenfold serial dilutions were plated onto −Leu media containing either glucose (indicated −HO, which represses expression of the HO endonuclease) or galactose (indicated +HO, which induces expression of the HO endonuclease). Mutant alleles were tested in parallel with wild-type YKU80 and vector controls.

FIG. 2.

yku80^tel mutants are defective for chromosome end protection. (A) Viability of yku80^tel est1-Δ double mutants generated by sporulation and dissection of the appropriate double heterozygous diploid (Table 1). Shown are 10-fold serial dilutions of equivalent numbers of cells taken directly from fresh dissection plates. (B) Viability of yku80^tel est1-Δ double mutants generated by plasmid shuffle. Plasmids (CEN TRP1) bearing yku80^tel mutant alleles were transformed into strain YVL1041 (yku80-Δ est1-Δ/pCEN URA3 EST1) (Table 1). Tenfold serial dilutions of equivalent numbers of cells were plated on −Trp 5-FOA media to evict the EST1 covering plasmid. (C) Analysis of telomeric end structure. Plasmids bearing yku80^tel mutant alleles were introduced into a yku80-Δ strain. The DNA was mock treated (−) or treated with E. coli exonuclease 1 (+). (Left panel [native]) Detection of telomeric G-strand overhangs (see Materials and Methods). (Right panel [denatured]) Detection of total telomeric DNA. The same gel following in-gel denaturation and rehybridization with the same oligomeric probe. (D) Western blot analysis. Plasmids expressing either untagged YKU80, YKU80_myc18, or yku80^tel_myc18 alleles were transformed into a yku80-Δ strain. Equivalent amounts of protein from whole-cell extracts were analyzed by Western blotting with an anti-myc antibody or with an anti-3′PGK antibody. The levels of Yku protein were normalized to the levels of PGK protein (data not shown). (E) Chromatin immunoprecipitation. Plasmids expressing either untagged YKU80, YKU80_myc18, or yku80-4_myc18 alleles were transformed into a yku80-Δ strain. Following formaldehyde cross-linking, protein-DNA complexes were immunoprecipitated with anti-myc antibodies. Telomeric DNA and high-copy-number nonspecific control sequence DNA (TyB) were detected by Southern blotting. The average of the results from 3 to 4 assays is shown. Each error bar indicates one standard deviation.

FIG. 3.

Ku has separable functions in telomere length regulation and telomere end protection (A) Telomere length analysis of yku80^tel mutants. Plasmids bearing yku80^tel mutant alleles were transformed into a yku80-Δ strain. A Southern blot of genomic DNA isolated after ∼65 generations of growth was probed to detect telomeric sequences. Independent isolates of each mutant allele were tested in parallel with wild-type YKU80 and vector controls. The four bands marked by arrows represent individual telomeres. (B) Analysis of telomeric end structure in a tlc1-Δ48 strain. Genomic DNA isolated from mid-log phase cultures of wild-type, tlc1-Δ48, and yku80-Δ strains. (Left panel [native]) Detection of telomeric G-strand overhangs. Indicated in parentheses is the native TRF signal/denatured TRF signal ratio normalized to that of the wild type. (Right panel [denatured]) Detection of total telomeric DNA.

FIG. 4.

Loss of Rif1 and Rif2 function does not rescue the growth defect of a yku80-Δ est2-Δ strain. Growth of isogenic haploid strains of the designated genotypes obtained by sporulation and dissection of YVL1071 (Table 1). Equivalent numbers of cells taken directly from the dissection plate were plated at a 10-fold serial dilution series.

FIG. 5.

yku80-5 through yku80-8 mutants are minimally impaired for telomere end protection but significantly impaired for telomeric silencing. (A) Viability of yku80^tel est1-Δ double mutants generated by plasmid shuffle. Plasmids (CEN TRP1) bearing yku80^tel mutant alleles, wild-type YKU80, and vector controls were introduced into strain YVL1041 (yku80-Δ est1-Δ/pCEN URA3 EST1) (Table 1). Tenfold serial dilutions of equivalent numbers of cells were plated onto −Trp 5-FOA media to evict the EST1 covering plasmid. (B) Serial streakouts following loss of the EST1 covering plasmid. The strains in panel A were streaked directly onto −Trp 5-FOA for single colonies (∼25 generations of growth following loss of the EST1 covering plasmid, left plate). Subsequently, single colonies were restreaked on −Trp (∼50 generations, right plate). (C) Viability of yku80^tel est1-Δ double mutants generated by sporulation and dissection of the appropriate double heterozygous diploid (Table 1). Tenfold serial dilutions of equivalent numbers of cells obtained directly from the dissection plate were plated. (D) Telomere length analysis of yku80^tel mutants 5 through 8. Plasmids bearing yku80^tel mutant alleles, wild-type YKU80, and vector controls were transformed into a yku80-Δ strain. After ∼65 generations, telomeric sequences were detected by Southern blot analysis of XhoI-digested genomic DNA. (E) Silencing of a telomere-located reporter URA3 gene is altered in yku80^tel strains. Plasmids bearing yku80^tel mutant alleles were transformed into strain YVL885 (yku80-Δ ppr1 ADE2-TEL_VR URA3-TEL_VIIL) (Table 1). Tenfold serial dilutions of cells were plated on −Trp media to determine plating efficiency (left), −Trp −Ura to evaluate the extent of URA3 derepression (middle), and −Trp 5-FOA to evaluate the extent of URA3 repression (right). Mutant alleles were tested in parallel with wild-type YKU80 and vector controls. (F) A telomere-located reporter ADE2 gene is stably derepressed in yku80^tel strains. Plasmids bearing yku80^tel mutant alleles and wild-type and vector controls were transformed into strain YVL885. Cells were plated on media selecting for the presence of the plasmid and limiting adenine to monitor ADE2 expression by colony color.

FIG. 6.

A subset of the yku80^tel mutants map to residues that are conserved between S. cerevisiae and humans. (A) Alignment of the S. cerevisiae Ku80 and human Ku80 proteins with secondary structure and fold recognition programs (see Materials and Methods). Alignment to the last 188 aa of the human Ku80 protein was not included, as this portion was lacking in the structural determination (61). Identical residues are shaded in black, and similar residues are shaded in gray. Positions of the yku80^tel missense mutants are indicated. The domains of the human Ku80 structure (61) are delineated by bars below the sequence. (B) Position of human Ku80 residues that correspond to the yku80^tel mutations mapped on a diagram based on crystal structure of Ku bound to DNA (61). Ku80 and Ku70 are shown in yellow and red, respectively, and DNA is in blue. The predicted positions of the yeast yku80^tel alleles are shown as cyan-colored space-filled residues.

FIG. 7.

A model for separable functions for Ku at the telomere. Ku associates with subtelomeric chromatin, where it influences the formation of heterochromatin. Independently, Ku associates with the chromosome terminus, where it mediates telomere length regulation via interactions with telomerase and telomere end protection via inhibition of an end-processing activity.