FIGURE 3:
The CENP-A nucleosome-binding domains of CENP-N and CENP-C have additive effects on stabilizing CENP-A nucleosomes in vitro. (A) MNase digestion analysis for complex AC and complex ANC. CENP-C426-537 was mixed with CENP-A nucleosome at a 2:1 ratio to form the complex AC. CENP-N1-289 was mixed with preformed AC complex at 2:1 ratio to form the ANC complex. DNA fragments after digestion were analyzed as in Figure 1A. (B) CENP-N1-289 and CENP-C426-537 showed additive effects in stabilizing the CENP-A nucleosome against dilution and heat. CENP-N1-289 or CENP-C426-537 was mixed with CENP-A nucleosome at molar ratios of 3:1. The buffer was adjusted to a final 20 mM Tris HCl, pH 7.5, 150 mM NaCl, 2% glycerol, 0.5 mM EDTA. Dilution and heat treatments were done as described in under Materials and Methods. Samples were kept at 4°C for 2 h before analysis by native PAGE. (C) Quantification of the percentage of stable nucleosomes under different nucleosome concentrations without heat (from the upper panel in B, n = 2. The raw signal (intensity of fluorescence signal at 647 nm) of nucleosome or complex after dilution (150 and 75 nM) was normalized to the signal from samples before dilution (at 300 mM NaCl). (D) The stabilizing effect of CENP-N and CENP-C on CENP-A nucleosome, as demonstrated by cryo-EM. Both CENP-N1-289 and CENP-C426-537 were mixed with CENP-A nucleosome at molar ratio 3:1 to form the ANC complex. Buffer was adjusted to the same condition for both samples. Concentration of both samples was 2.5 µM. Red boxes indicate nucleosome-shaped particles. Scale bar = 50 nm. Blue box highlights the area from the left micrograph. The intact particles were counted, and the numbers are listed in Table 1.