Mutations of the Yku80 C terminus and Xrs2 FHA domain specifically block yeast nonhomologous end joining

Abstract

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.

FIG. 1.

NHEJ yeast two-hybrid screen. (A) All NHEJ gene constructs were cloned by gap repair into both pOBD2 (bait) and pOAD (prey) vectors. Two independent bait isolates were arrayed in a 96-well microtiter dish. Two independent prey isolates were pooled and mated against the bait array to generate arrays of diploids containing paired bait and prey clones. (B) Schematic summarizing the two-hybrid results. Double-headed arrows indicate interactions that were detected by reciprocal bait-prey pairings. Single-headed arrows indicate interactions that were detected in only one orientation, with the arrow pointing to the prey. Arrows point to the domain responsible for the interaction as determined from the pattern of positive clones. Shading is used to indicate the previously known stronger interactions. Note that some proteins are drawn in reverse. (C) Plasmids mediating novel interactions were isolated, sequenced, and transformed into PJ694-α. Two-hybrid interactions were assessed by spotting to −His and −Ade plates and assaying for lacZ expression by measuring β-galactosidase activity.

FIG. 2.

Ku80 alignment and suicide deletion assay. (A) Comparison of human and yeast Ku80 showing conserved VWA and β-barrel core domains but little conservation of the C termini. (B) Alignment of the fungal Ku80 C terminus demonstrates a cluster of conserved amino acids between residues 605 and 619. Species shown are as follows: Sc, Saccharomyces cerevisiae; Sm, Saccharomyces mikatae; Sb, Saccharomyces bayanus; Sp, Saccharomyces paradoxus; Sku, Saccharomyces kudriavzevii; Skl, Saccharomyces kluyveri; Scas, Saccharomyces castelli; Cg, Candida glabrata; Ag, Ashbya gossypii; Kl, Kluyveromyces lactis. Numbers indicate S. cerevisiae Yku80 residues. Black shading, residues that are identical in all proteins; light gray shading, residues identical to S. cerevisiae; dark gray shading, conservative substitutions. (C) HO(+2) suicide deletion assay. Galactose-induced expression of the HO endonuclease results in deletion of the HO coding sequence and a DSB in the ADE2 marker. DSB repair by NHEJ allows cell survival, with repair in the +2 frameshift register resulting in Ade⁺ cells. The principal joint responsible for Ade⁻ and Ade⁺ colonies is shown.

FIG. 3.

Truncation of the Yku80 C terminus results in NHEJ-specific defects. (A) Effects of Yku80 C-terminal truncations on total NHEJ repair by HO suicide deletion. The yku80Δ605-629, Δ591-629, and Δ577-629 mutants showed a modest twofold reduction in total NHEJ. (B) Effects of Yku80 C-terminal truncations on +2 imprecise NHEJ by HO suicide deletion. Data are plotted as the ratio of Ade⁺ to total colonies. The yku80Δ605-629, Δ591-629, and Δ577-629 yeast strains showed a ninefold reduction in +2 imprecise events. No Ade⁺ events were recovered with the yku80Δ strain. Error bars indicate standard deviations for at least three replicates. (C) Haploid strains containing the yku80 mutations and the URA3-TPE allele were spotted to SD complete, SD-Ura, and SD 5-FOA plates. Complete plates were incubated at 37°C. Strains were also quantitatively plated to media with and without 5-FOA to determine the percentage of FOA-resistant cells (% FOA^R). Unlike the yku80Δ and yku80Δ552-629 yeast strains, the yku80Δ605-629, Δ591-629, and L609A yeast strains did not show compromised growth at 37°C and were also able to epigenetically silence the URA3 marker.

FIG. 4.

The Xrs2 FHA domain and the Yku80 C terminus act synergistically during NHEJ. (A) Genotypes of YW1276 derivative strains used in this figure. Letters from this table are used to designate strains in panels B to F. Strain I (xrs2Δ yku80Δ) displayed rapid senescence and could not be used for further experiments (indicated by shading). (B and C) The yku80Δ605-629 xrs2ΔFHA double mutant (strain E) demonstrates synergistic inhibition of precise NHEJ (B) and +2 imprecise NHEJ (C) compared to the corresponding single mutants (strains B and D) in the HO(+2) suicide deletion assay as described in the legend to Fig. 2. (D) Double-mutant strain E is not temperature sensitive or hypersensitive to camptothecin (10 μg/ml), hydroxyurea (30 mM), or a 30-min exposure to 0.3% MMS. (E) Telomeric Southern blotting showing that the yku80Δ605-629 and xrs2ΔFHA mutants have wild-type telomere length. Y′-type telomeres are indicated. (F) Strains B to H are deficient in recircularization of a plasmid with a DSB in the LEU2 marker following transformation. Error bars indicate standard deviations for at least three replicates.

FIG. 5.

Identification of conserved Yku80 C-terminal residues mediating NHEJ. Residues 606 to 616 were replaced individually with alanine, and the total (A) and +2 imprecise (B) NHEJ efficiencies were determined as for Fig. 2. yku80-L609A, -L612A, and -L613A mutants demonstrate total NHEJ and +2 NHEJ defects similar to those of yku80Δ605-629 in the XRS2 wild-type strain. Deletion of the Xrs2 FHA domain synergistically impaired total (C) and +2 imprecise (D) NHEJ in the leucine mutants and uncovered lesser defects in other residues. ND, not done; mutations were not tested in the xrs2ΔFHA background. Error bars indicate standard deviations for at least three replicates.

FIG. 6.

The NHEJ critical region of theYku80 C terminus. (A) The crystal structure of the human Ku70/80 heterodimer (light gray) bound to DNA (dark gray) reveals that the C terminus of Ku80 is oriented toward the DSB terminus. The C-terminal residue of the partial Ku80 protein is shown in black. (B) Schematic representation of three classes of Ku80 C termini. The α-helical bundle seen in human Ku80 NMR structures is schematized in black. The DNA-PKcs-interacting region is shown in dark gray. The putative Dnl4-interacting region is shown in light gray. A possible homologous region in M. grisea is shown in white. Numbers indicate amino acid positions. (C) Helical wheel predictions for the three regions indicated in panel B.