Figure 4.

DNA binding and nucleoprotein condensate formation by the RM-TDB domain. (A) Representative EMSA of binding to a 150-bp DNA substrate. (B) Comparison of DNA binding (150-bp substrate) by RM-TDB complexes composed of different fragments of Rec114 and Mei4. Error bars indicate mean ± range (two replicate experiments) or mean ± SD (three replicates). C₅₀ values were 90 nM ± 20 nM for Rec114_N–Mei4_1–90 (mean ± range), 90 nM ± 30 nM for Rec114_C–Mei4_N (mean ± SD), and 110 nM ± 30 nM for Rec114_386–428–Mei4_N (mean ± range). The EMSA for Rec114_388–428–Mei4_13–43 was conducted once (C₅₀ of ∼100 nM). (C) AFM images of 200 nM Rec114_C–Mei4_N in the absence of DNA or presence of 1 ng/µL supercoiled pUC19 plasmid DNA, relaxed circular pHOT1 plasmid DNA, or 1000-bp linear DNA. (D) Confocal images of condensates formed by 450 nM fluorescently labeled (LD650) Rec114_C–Mei4_N and 25 nM 1000-bp Cy3 DNA. (E,F) Quantification of the radius of central cores (E) and thickness of haloes (F) of the condensates from confocal images (N = 20). Dashed lines indicate the expected contour length of free DNA (0.383 µm). (G) AFM images of 1 ng/µL 1000-bp linear DNA in the absence (top) or presence (bottom) of 70 nM Rec114_C–Mei4_N. Examples of DNA molecules with bound protein (dashed boxes) are shown in zoomed insets at the right. (H) DNA contour lengths of free DNA (blue points) and protein-bound DNA (with N_p indicating the number of protein particles per DNA molecule). Error bars indicate mean ± SD. Free DNA, 358 nm ± 16 nm (N = 474 DNA molecules); N_p = 1 particle bound, 290 nm ± 48 nm (N = 16 DNA molecules); N_p = 2 particles bound, 272 nm ± 41 nm (N = 28 DNA molecules); N_p = 3 particles bound, 283 nm ± 34 nm (N = 21 DNA molecules); N_p > 3 particles bound, 240 nm ± 38 nm (N = 5 DNA molecules). The pairwise P-values are from unpaired two-tailed Student's t-tests. The group P-value for different numbers of protein particles bound is from a Kruskal–Wallis test.