Mechanism of MRX inhibition by Rif2 at telomeres

Abstract

Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5' end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5' resection.

Fig. 1. Rif2 N-terminal region inhibits NHEJ at telomere.

A Schematic representation of S. cerevisiae Rif2 and sequence conservation of Rif2 and Orc4 N-terminal region in Saccharomycetales species^,,,. In some post-WGD (whole genome duplication) Saccharomycetacae species, Rif2 and Orc4 are syntenic and only Rif2 possesses a BAT motif. In other Saccharomycetacae species, the motif is found in Orc4. Core residues of the BAT motif are also present in Orc4 N-terminal region in non-Saccharomycetacae species of the Saccharomycetales order. The alignment was extended downstream towards a conserved motif, which might correspond to a Rap1-binding module (RBM). Alignment of the full-length proteins is shown in Supplementary Fig. 1. (Sequence Accession numbers: Rif2: Saccharomyces cerevisiae: Q06208, Kazachstania africana: XP_003954885.1, Naumovozyma_castellii: XP_003677686.1, Orc4: Saccharomyces cerevisiae: P54791, Kazachstania africana: XP_003955485.1, Naumovozyma_castellii: XP_003674441.1, Tetrapisispora_phaffii: XP_003686307.1, Vanderwaltozyma_polyspora: XP_001643535.1, Zygosaccharomyces_rouxii:XP_002496500.1, Torulaspora_delbrueckii: XP_003683125.1, Kluvyeromyces_lactis: XP_452959.1, Eremothecium_gossypii: NP_983126.1, Cyberlindnera fabianii: ONH65289.1, Debaryomyces hansenii: XP_459748.2, Clavispora XP_002615148.1, Candida dubliniensis XP_002420502.1, Candida auris: PIS52278.1). B Fusing Rif2 N-terminal region to Rap1 C-terminal end (RAP1-RIF2_1-60) protects telomeres against NHEJ-dependent fusions in cells lacking Rif2 and Sir4. Fusions between X and Y′ telomeres were detected by semi-quantitative PCR (upper panels: 30 and 26 cycles, lower panels: 34 and 30 cycles). Quantification of the amplified products indicated for each lane (ng). Serial 4-fold dilution of the template DNA from rif2∆ sir4∆ and tel1∆ rif2∆ sir4∆ cells provides an estimation of the method sensitivity. Rarer fusions are amplified as discrete bands. Experiment reproduced three times.

Fig. 2. Rif2 BAT motif inhibits NHEJ at broken ends.

A I-SceI assay used to estimate NHEJ efficiency. Two inverted I-SceI sites are inserted at the endogenous URA3 gene. Most survivors to continuous I-SceI expression have eliminated the I-SceI sites by fusing the distal broken ends. B NHEJ inhibition by Rap1 C-terminal domain and Rif2 N-terminal region targeted at broken ends (lif1∆: NHEJ-deficient cells). Means from independent cell cultures. C Increasing distances between the broken end and the Gal4 binding sites decrease NHEJ inhibition by Rif2. D Rif2 N-terminal truncations impacting the ability to inhibit NHEJ at broken ends. Gal4_DBD and Gal4_DBD-Rif2_fragments expressed from a centromeric plasmid. E Rif2 mutations impacting its ability to inhibit NHEJ at broken ends. Gal4_DBD and Gal4_DBD-Rif2_1-60 expressed from an integrated plasmid. Gal4_DBD-Rif2_1-395 expressed from a centromeric plasmid. F The rif2-F8A mutation exposes telomeres to NHEJ in cells lacking Sir4 (fusions between X and Y′ telomeres). Experiment reproduced three times. G K. lactis Orc4 expression complements Rif2 loss for NHEJ inhibition by Rap1 C-terminal domain in S. cerevisiae.

Fig. 3. Rif2 BAT blocks 5′ resection and MRX complex presence at broken ends.

A Rif2 N-terminal region stabilises broken ends. Left panel: the stability of I-SceI-induced broken ends with 5 Gal4 sites determined by Southern blot in G1 and G2/M arrested cells. Right panel: quantification of the uncut and cut signals normalised to the control ADE1 signal. Means from independent samples. Experiment reproduced three times. B Mre11 and Xrs2 presence at I-SceI-induced broken ends with 5 Gal4 sites determined by ChIP in G1 arrested cells. Quantification of immunoprecipitated DNA (IP) relative to the input DNA (IN). Means from independent samples. Quantification of I-SceI cleavage efficiency in Supplementary Fig. 4D.

Fig. 4. Rif2 BAT interacts with Rad50 ATPase Heads in vivo.

A 2-Hybrid interactions between Rif2 N-terminal region and full-length Rad50 in WT cells and in cells lacking Mre11 and Xrs2 (− no growth on plates supplemented with 3-AT, + growth on plates supplemented with 3-AT). A: slow growth in MRX-defective cells. B 2-Hybrid interactions between Rif2 N-terminal region and full-length Rad50 or Rap1 (fragment 366–827) in WT cells (+/- slow growth on plates supplemented with 3-AT). C Representation of full-length Rad50 and of the Rad50_∆CC fragment lacking the coiled-coil arm. D 2-Hybrid interactions between Rif2 N-terminal region and Rad50 ATPase Head in WT cells and in cells lacking the endogenous Rad50. E GST pull-down interaction between Rif2 N-terminal region and Rad50 ATPase Head. Experiment reproduced three times.

Fig. 5. Identification of Rif2-resistant Rad50 mutants through a genetic screen.

A Schematic representation of the chromosome fusion capture assay used to identity Rif2-resistant Rad50 mutants. The loss of chromosome 6 centromere (CEN6) generates a lethal acentric chromosome unless chromosome 6 fused to another chromosome. The Cre recombinase is expressed from a galactose-inducible promoter. Cre-induced CEN6 loss generates a functional LEU2 gene at the CEN6 locus. The 5′ end of RAD50 ORF (−105 to +996) was mutagenized by PCR using the Taq polymerase. The mutant library was transformed in cells lacking Sir4 and Rad50. 300 individual transformants were patched to saturation on rich medium prior to being replicated on synthetic medium with galactose (2%) and lacking leucine to identify clones with increased survival rate to CEN6 loss. B Quantification of the survival to CEN6 loss in the 13 rad50 mutants identified in the screen (m1–m13). Cells were grown to saturation prior to plating on synthetic medium with galactose (2%) and lacking leucine. Colonies were counted after 5d at 30 °C. Means from independent cell cultures. Mean telomere length from Supplementary Fig. 5. C NHEJ inhibition by Rif2 BAT motif at I-SceI-induced broken ends in selected rad50 mutants (single I-SceI site assay (Supplementary Fig. 2B)). Means from independent cell cultures. D Position of rad50-m7 (K81) and rad50-m13 (T22, I43, I93) mutated residues on a model structure of S. cerevisiae Rad50 N-terminal Head domain (1–189, obtained from Chaetomium thermophilum Rad50 structure as template (PDB:5DAC)).

Fig. 6. Identification of Rad50 residues essential for BAT function and interaction.

A Position of the eight residues (red) selected with CABSdock as potentially interacting with Rif2 BAT. B 2-Hybrids interactions between mutant full-length Rad50 and Rif2 N-terminal region or full-length Mre11. C GST pull-down interaction between Rif2 N-terminal region and Rad50 ATPase Head. Experiment reproduced three times. D NHEJ inhibition by Rif2 BAT motif at I-SceI-induced broken ends in rad50 mutants (single I-SceI site assay (Supplementary Fig. 2B)). Means from independent cell cultures. E Impact on telomere length of mutants rad50-K6A and rad50-K81E in WT cells and in cells lacking Rif1 or Rif2 (Southern blot, Y′ probe, XhoI digest). Experiment reproduced three times.

Fig. 7. Predicted impact of BAT binding on the Rad50–Mre11 complex and model for MRX inhibition by Rif2 at telomeres.

A Models of S. cerevisiae Rad50–Mre11 complex in the ATP-bound resting state (left) and in the DNA-bound active state (right). Residues K6, K81 and I93 highlighted in red. Bottom panel: envelope of the BAT core peptide (Rif2 residues 4-14, in brown) belonging to the HADDOCK cluster 1 shown after superimposition of the Rad50 structures of the clusters on that of the DNA-bound Rad50–Mre11 complex model. B Model for telomere protection by Rif2 at telomeres (Rad50 orange/yellow, Mre11 teal/light blue, Rap1 grey, Rif2 pink, BAT and BAT interacting region on Rad50 red). For simplicity, Xrs2 and other factors present at broken ends and at telomeres are not represented.