Functional and molecular insights into the role of Sae2 C-terminus in the activation of MRX endonuclease

Abstract

The yeast Sae2 protein, known as CtIP in mammals, once phosphorylated at Ser267, stimulates the endonuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex to cleave DNA ends that possess hairpin structures or protein blocks, such as the Spo11 transesterase or trapped topoisomerases. Stimulation of the Mre11 endonuclease by Sae2 depends on a Rad50-Sae2 interaction, but the mechanism by which this is achieved remains to be elucidated. Through genetic studies, we show that the absence of the last 23 amino acids from the Sae2 C-terminus specifically impairs MRX-dependent DNA cleavage events, while preserving the other Sae2 functions. Employing AlphaFold3 protein structure predictions, we found that the Rad50-Sae2 interface involves not only phosphorylated Ser267 but also the phosphorylated Thr279 residue and the C-terminus of Sae2. This region engages in multiple interactions with residues that are mutated in rad50-s mutants, which are known to be specifically defective in the processing of Spo11-bound DNA ends. These interactions are critical for stabilizing the association between Sae2 and Rad50, thereby ensuring the correct positioning of Mre11 in its active endonucleolytic state.

Graphical Abstract

Figure 1.

Search for sae2 mutants defective in hairpin resolution. (A) Schematic representation of the lys2::Alu IR and lys2-Δ5′ ectopic recombination system. (B) Exponentially growing cultures were serially diluted, and each dilution was spotted out on SC medium with or without lysine and on yeast extract, bactopeptone, and glucose (YEPD) plates with or without CPT or phleomycin. (C) Recombination frequency of strains with the lys2::Alu IR and lys2-Δ5′ ectopic recombination system. The rate of Lys⁺ recombinants was derived from the median recombination frequency. The mean values of three independent experiments are represented with error bars denoting s.d. ***P< 0.005 (Student's t-test). (D) Percentage of sporulation. Diploid cells homozygous for the indicated mutations were induced to enter meiosis. The mean values of three independent experiments are represented with error bars denoting s.d. ***P< 0.005 (Student's t-test).

Figure 2.

The sae2-ΔC23 mutation does not impair resection of an endonuclease-induced DSB. (A) Western blot analysis with an anti-Myc antibody of protein extracts prepared from exponentially growing cells. The same amount of extracts was stained with Coomassie Blue as loading control. (B) Co-IP. Protein extracts from exponentially growing cells were analyzed by western blotting with anti-HA and anti-Myc antibodies either directly (Total) or after IPs of Rad50-HA with an anti-HA antibody. (C) DSB resection. JKM139 derivative strains were transferred from YEPR to YEPRG at time zero. SspI-digested genomic DNA was hybridized with a single-stranded MAT probe that anneals with the unresected strand. 5′-3′ resection produces SspI fragments (r1 to r6) detected by the probe. (D) Densitometric analysis of the resection products. The mean values of three independent experiments as in (C) are represented with error bars denoting s.d. (E, F) Exponentially growing cells were serially diluted and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or HU at the indicated concentrations.

Figure 3.

The sae2-ΔC23 mutation enhances Mre11 and Tel1, but not Rad9 association with DSBs. (A–C) Western blot analysis with anti-Myc or anti-HA antibodies of protein extracts prepared from exponentially growing cells. The same amount of extracts was stained with Coomassie Blue as loading control. (D–F) ChIP and qPCR. Exponentially growing YEPR cell cultures were transferred to YEPRG to induce HO expression, followed by ChIP analysis of the recruitment of the indicated proteins at 600 bp from the HO-cut site. In all diagrams, ChIP signals were normalized to the corresponding input signal for each time point. The mean values of three independent experiments are represented with error bars denoting s.d. ***P< 0.005 (Student's t-test). (G) Adaptation assay. YEPR G1-arrested cell cultures were plated on galactose-containing plates (time zero). At the indicated time points, 200 cells for each strain were analyzed to determine the frequency of large, budded cells (two cells) and cells forming microcolonies of more than two cells (>2 cells). (H) Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero to induce HO. Western blot analysis of protein extracts was performed with anti-Rad53 antibodies. (I) Co-IP. Protein extracts prepared from exponentially growing cells were analyzed by western blotting with anti-HA (Rad9) and anti-Rad53 antibodies either directly (Total) or after IP of Rad9-HA with an anti-HA antibody. *indicates a cross-hybridization signal.

Figure 4.

AlphaFold-based models for Sae2 C-terminal region binding to Rad50 and Mre11. (A) Cartoon representation of the 202–345 amino acid region of Sae2 (in magenta) phosphorylated at S267 and T279 residues, as modeled by AlphaFold3 server, together with Rad50 (in gold and with the aa 214–1107 coiled-coil region deleted), and Mre11 (aa 1–416, in blue) was superimposed on the AlphaFold2 model of Sae2 S267E (in pink) and Rad50 (in light yellow) for comparative analysis. (B) Model of the cutting state for Mre11–Rad50–Sae2 complex bound to DNA. The proteins are represented as cartoons with transparent surfaces, while the DNA molecule is depicted as a cartoon. Mre11 subunits are displayed in different shades of blue, Rad50 in yellow and light orange, and Sae2 in magenta.

Figure 5.

AlphaFold3-based model for Sae2 C-terminal region binding to Rad50. (A) Cartoon representation of the Sae2 202–345 amino acid region containing the phosphorylated Sae2 S267 and T279 residues, as modeled by the AlphaFold3 algorithm, together with Rad50, from which the amino acid 214–1107 coiled-coil region has been omitted, and Mre11 (aa 1–416). (B,C) The polar interactions between the Sae2 chain and the Rad50 chain are represented as a cartoon. Residues involved in contacts are represented as sticks and H-bonds are represented as dashed black lines. Nitrogen atoms are colored in blue, oxygen in red, and hydrogen in white. The view is rotated by 90° on the Y-axis from panel B to panel C.

Figure 6.

The binding site of the Sae2 C-terminus and relative mutations. (A) Recombination frequency of strains with the lys2::Alu IR and lys2-Δ5′ ectopic recombination system. The rate of Lys⁺ recombinants was derived from the median recombination frequency. The mean values of three independent experiments are represented with error bars denoting s.d. *P< 0.05, ***P< 0.005 (Student's t-test). (B) Exponentially growing cultures were serially diluted, and each dilution was spotted out on YEPD plates with or without CPT. (C) Details of the binding site of the C-terminus of Sae2 with Rad50. The polar contacts of R286 and R344 of Sae2 and D19 and E21 of Rad50, targeted for mutation and highlighted in pink (Sae2) and orange (Rad50), are shown as black dashed lines. (D) As in A. (E) Exponentially growing cultures were serially diluted, and each dilution was spotted out on YEPD plates with or without CPT.

Figure 7.

Targeted mutagenesis and compensatory effects. (A) Details of the salt bridge between Sae2 phosphorylated residue T279 and Rad50 K6. (B) Recombination frequency of strains with the lys2::Alu IR and lys2-Δ5′ ectopic recombination system. The rate of Lys⁺ recombinants was derived from the median recombination frequency. The mean values of three independent experiments are represented with error bars denoting s.d. ***P< 0.005 (Student's t-test). (C) Details of the complex network of polar interactions involving Sae2 D285, R273, K288 and E281, and Rad50 R20. (D) As in B. (E) Details of the polar interactions between the Sae2 K296 residue and Rad50 N18 and E73. (F,G) As in B. Residues involved in contacts are represented as sticks and polar interactions are represented as dashed black lines. Nitrogen atoms are colored in blue, oxygen in red, and hydrogen in white.