(a) DMH3-H4 is primarily a dimer, even under conditions that favor the tetrameric state of WTH3-H4. Gel filtration of WTH3-H4 and DMH3-H4 in refolding buffer (2M NaCl). (b) tCAF-1 binds equally well to WT or DMH3-H4, determined by fluorescence polarization (Kd ~0.5–0.7 nM). Error bars show the S.E.M. from three independent measurements. (c) van Holde-Weischet analysis of SV-AUC experiments performed with FL CAF-1 and H3-H4 (wild type and DMH3-H4), combined in a 1:1 or 1:2 molar ratio of CAF-1 to H3-H4 dimer. An increased S-value upon addition of a second equivalent of H3-H4 dimer (but not with a second DM-H3H4) indicates that a (H3–H4)2 tetramer may form on CAF-1. (d) SDS PAGE of histones before and after the chemical crosslinking to form XL(H3-H4)2. (e) tCAF-1 binds equally well to WT or XL(H3-H4)2, as determined by fluorescence polarization (Kd ~0.5–0.9 nM). Error bars show the S.E.M. from three independent measurements. (f) FRET-based Job plot experiment, using FL CAF-1 and either WT or DMH3-H4, shows a single binding event for both histone complexes (protein concentration was kept at 150 nM, where WT histones are dimeric). With XL(H3-H4)2, we observed a stoichiometry of two CAF-1 complexes per tetramer. Error bars show the SEM of two independent titrations. (g) Size-exclusion chromatography (S200 10/300 GL) and SDS PAGE of tCAF-1•WTH3-H4 complex. The buffer contained 30 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP. (h) Schematics to show the binding stoichiometry of CAF-1 to different H3-H4 isoforms.