Fig 1: Noncanonical DNA interaction surface and phosphorylation of TRF1 crucial for nucleosome binding.(A) Interactions between the two TRF1 Myb domains with teloNCP. Dashed box and circle indicate histone H3-Myb2 and DNA-Myb2 interactions, respectively. (B) Left: Close-up view of the interaction between the phosphorylated C-terminal residues of TRF1 Myb2 domain with histone H3 N-terminal tail in the TRF1core-teloNCP structure. Right: Close-up view of the same region of histone H3 interacting with DNA in the apo-teloNCP structure. Black circles highlight the observation that the phosphate group of S434 occupies the same position as a DNA backbone phosphate. (C) Close-up view showing noncanonical DNA interactions made by helix 1 of Myb2. (D) Cryo-EM density of the phosphorylated C-terminal residues of TRF1 Myb2 domain. (E) Phos-tag gels of the untreated and ?-phosphatase (? PPase) treated TRF1core samples combined with immunoblotting using TRF1, TPP1, and TIN2 antibodies (a-TRF1, a-TPP1, and a-TIN2). (F) Sequence alignment of TRF1 Myb domains from various mammalian species and human TRF2 Myb domain. The hexagonal dots and stars underneath the sequence denote residues involved in DNA interaction within the noncanonical DNA surface on Myb2 (C) and residues involved in interactions with histone H3 (B), respectively. (G) EMSAs showing titration of purified wild-type (WT) and mutant TRF1core complexes against teloNCP. Experiments were performed in triplicate. (H) Quantification of EMSA experiments shown in (G). In the left, we plotted percentages of unbound teloNCPs as a function of protein concentration in the EMSA reaction of the wild-type and each mutant complex. The right table shows the concentration of each TRF1core complex at which 50% of teloNCP remains unbound as determined from the graphs. Error bars at each concentration point are the SEM obtained from the three replicates.