Figure 1. Kinase-independent function of Rad3^ATR-Rad26^ATRIP contributes to telomere maintenance in the absence of the Nbs1-Tel1^ATM interaction domain of Nbs1.

(A) Tel1^ATM-MRN (Rad32^Mre11-Rad50-Nbs1) and Rad3^ATR-Rad26^ATRIP represent two redundant pathways essential for telomere maintenance in fission yeast. (B) Schematic representation of Nbs1 protein expressed in nbs1⁺ and nbs1-c60Δ. Conserved motifs and functional domains [5],[27] are indicated. (C) Schematic representation of various Rad3 constructs tested. Amino acids 1477–2386 are deleted in rad3-kdΔ, while amino acids 1–2363 are deleted in rad3Δ. Conserved motifs and functional domains are indicated [26],[58]. The Leucine zipper (LZ) and putative PCNA-interaction site (P-site) were previously implicated in protein-protein interaction [59]. (D) Characterization of telomere status by pulsed-field gel electrophoresis (PFGE) for indicated strains. Cells with defects in telomere maintenance show C+M and I+L bands, corresponding to the fused telomeric fragments. Bottom, NotI restriction map of fission yeast chromosomes, shown with telomeric C, I, L, and M fragments marked as black boxes. (E) Rad3-kdΔ forms a stable complex with Rad26 in vivo. Anti-myc immunoprecipitation was performed using extracts from strains expressing YFP-Rad26 and either wild-type myc-Rad3 or myc-Rad3-kdΔ, and co-immunoprecipitated Rad26 was visualized by anti-GFP western blot. (F) Two-hybrid assays of S. pombe Rad26 with full-length Rad3 (Rad3⁺), C-terminal truncation of Rad3 (Rad3-kdΔ), or the N-terminal 191 amino acids of Rad3. A positive two-hybrid interaction is identified by growth on -His plates.