(A) Fluorescence polarisation assay showing that a fluorescent peptide (Flu-GYGVEGDHS(pS)D(pT)EEEVVS) incorporating the single SDT site in Mdb1, the fission yeast homologue of MDC1, does not interact with Rad4-BRCT3,4 as has been suggested for the SDT sites in human MDC1 with the homologous TOPBP1-BRCT4,5 (Leung et al., 2013; Wang et al., 2011). The Mdb1-SDT site peptide does, however, interact with high-affinity with the S. pombe homologue of NBS1. (B) Mdb1 contains a sequence motif with a potential phosphorylation site at Thr 113 that corresponds closely to the consensus for binding to Rad4/TOPBP1-BRCT2. (C) Fluorescence polarisation assay of an Mdb1-derived phosphopeptide binding to the Rad4-BRCT1,2 segment. The Mdb1-pT113 peptide binds with high affinity to the wild-type BRCT1,2 but only weakly when a disruptive mutation is introduced into the phosphate-binding site of BRCT2, but is largely unaffected by comparable mutations in BRCT1. All detectable bindings are lost when both BRCT domains are mutated. (D) Crystal structure of Mdb1-pT113 peptide bound to the BRCT2 domain within the Rad4-BRCT1,2 module. (E) Closeup of interactions. As in other complexes with BRCT2, the single hydrophobic residue at −3 relative to the phosphorylated residue binds into the hydrophobic pocket, with the main chain for the −2, –3 and −4 residues adopting an extended conformation, rather than the tight turn seen in interactions with BRCT1.