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^21,22,28,30. 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 order³⁰. The alignment was extended downstream towards a conserved motif, which might correspond to a Rap1-binding module (RBM)²². 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.