Fig. 2: Direct observation of BRCA2 binding to telomere G4 at the single-molecule level. | Nature Communications

Fig. 2: Direct observation of BRCA2 binding to telomere G4 at the single-molecule level.

From: Dynamic interaction of BRCA2 with telomeric G-quadruplexes underlies telomere replication homeostasis

Fig. 2

a TelG5, TelG5TTT, and TelG5T2G4 probes (10 nM) were incubated with BRCA2OB at the following concentrations: 0, 5, 10, 20, 50, 100, 200, 500, and 1000 nM (left). The Kd values were analyzed by curve-fitting using nonlinear regression (mean ± s.d.) (Right). TelG5, Kd = 4.4 μM; TelG5TTT, Kd = 456 nM; TelG5T2G4, Kd = 1.4 μM. The result is from more than three independent experiments. b Purified BRCA2OB and POT1OB were subjected to EMSA in the presence of 100 mM KCl with G4-forming telomere variant repeats (TVRs); TelG5, TelG5TTT, TelG5T2G4, and unfolded mutant TelG5GAG. Human POT1 and the POT1 OB-fold domains (POT1OB) are illustrated at top right. The OB-folds and TPP1 binding domain are marked. Bar graph, binding intensity relative to TelG5 in EMSA (mean ± s.e.m.). The experiments were repeated at least four times independently. c Schematic drawing of the single-molecule FRET (smFRET) assay with dual-labeled TelG5 and BRCA2OB. The G4 conformational dynamics between the unfolded (UF), non-parallel (NP), and parallel (P) folded conformations, and BRCA2OB-bound (BD) state are shown. d CD (circular dichroism) spectra of the TelG5 without salt (black), in 10 mM KCl (yellow, dashed), and 100 mM KCl (orange). e A smFRET histogram of TelG5 with three Gaussian fits (red) of the UF, NP, and P conformational peaks in the presence of 100 mM KCl. The blue-streaked curve represents the sum of the Gaussian fits. f Representative time trajectory of the TelG5 exhibiting dynamic transitions between UF, NP, and P conformations in 100 mM KCl. g A smFRET histogram of TelG5 incubated with 1 μM of BRCA2OB. The BRCA2OB-bound (BD) state is indicated by the black curve filled with semi-transparent blue. h Differential density histogram obtained by subtracting the molecular density of FRET efficiency for TelG5 alone (e) from that in the presence of BRCA2OB (g). i Representative time trajectory of TelG5 in the presence of 1 μM BRCA2OB. The BD state upon binding of BRCA2OB is marked (orange broken line).

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