Donham DC , Scorgie JK , Churchill ME .

R01 GM079154 NIGMS NIH HHS , R01 GM079154-01A1 NIGMS NIH HHS , R01 GM079154-02 NIGMS NIH HHS , R01 GM079154-03 NIGMS NIH HHS , R01 GM079154-04 NIGMS NIH HHS

	Figure 1. Asf1-assisted exchange of histone dimers onto DNA. (A) Tetrasome and disome formation in the absence of yAsf1. The 601, 5SDNA and a non-positioning sequence (NPS) 80 bp DNA fragments at 0.4 µM concentration were incubated with a 2-fold excess of H3/H4*FM or H3K56Q/H4*FM dimers prior to analysis by non-denaturing PAGE. The gel was scanned to obtain the H3/H4*FM fluorescence before the gel was stained with SYBR Green I nucleic acid stain and then rescanned, as described in Supplementary Figure S2. The DNA and histones are shown in green and the position of each species is indicated with an arrow (top panel). The products were analyzed by non-denaturing PAGE and scanned for FM fluorescence. The data from at least three independent experiments were quantitated and are presented in graphical form (bottom panel). (B) Effect of excess of yAsf1 on the formation of tetrasomes and disomes in the presence of an excess of histones. The molar ratios of each component of the mixture are indicated above the non-denaturing PAGE image, which was produced as described in (A). (C) Effect of prior addition of Asf1 on tetrasome and disome formation. H3/H4*FM histones at 0.8 µM were incubated in the absence and presence of increasing concentrations of unlabeled yAsf1 (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, or 2.0 µM) for 30 min at 20°C prior to addition of 0.4 µM concentration 80 bp DNA fragments of 5SDNA. After further incubation for 60 min at 20°C the products were analyzed by non-denaturing PAGE, scanned for FM fluorescence and the data from between three and six independent experiments are presented in graphical form.
	Figure 2. yAsf1 does not bind to or dissociate tetrasomes by itself. (A) Titration of yAsf1* into 0.4 µM 5SDNA or 601 80 bp tetrasomes. yAsf1* was added at 0, 0.4, 0.8, 2 and 4 µM. The images show the fluorescence of yAsf1* in red and histones H3/H4*FM and SYBR Green I stained DNA in green. (B) Titration of yAsf1 into 0.4 µM 5SDNA or non-positioning sequence (NPS) 80 bp tetrasomes formed with H3K56Q/H4*FM histones. yAsf1* was added at 0, 0.4, 0.8, 2 and 4 µM. The images were produced as in (A).
	Figure 3. Effect of HMGB1 or supercoiled DNA on tetrasome dissociation by Asf1. (A) Effect of HMGB1 on stability of tetrasomes in the presence of yAsf1. HMGB1 was added to give a final concentration of 0, 0.4, 0.8, 2 and 4 µM with 0.4 µM 5SDNA and 601 tetrasomes. The gels were scanned and processed as in Supplementary Figure S1, with fluorescence of H3/H4*FM and SYBR Green I stained DNA shown in green, and yAsf1* in red. (B) DNA competition assay. Supercoiled or linear 5SDNA or 601 plasmid DNA was added at 0.02 µM concentration to tetrasomes in the absence or presence of yAsf1. The images were produced as in (A).
	Figure 4. Analytical ultracentrifugation of H3/H4 at different ionic strengths. (A) Sedimentation velocity AUC analysis of H3/H4. The normalized c(s) distribution curves calculated from the raw data using SEDFIT (44) are shown for H3/H4 at 10 µM (dimer in red) and carbonic anhydrase (CA in black) in buffers containing 2 M NaCl (solid line) and 150 mM NaCl (dotted line), respectively. (B and C) Sedimentation equilibrium concentration profiles of H3/H4 were measured in buffers with 150 mM (B) and 2 M (C) NaCl concentrations, respectively. The absorption profile was recorded at 280 nm at three rotor speeds each: 15 000 rpm (red circle), 25 000 rpm (green triangle), and 30 000 rpm (blue square). The continuous line is the non-linear least-squares global fit to a single exponential function, with single species having a molecular masses shown in Table 2, as analyzed with SEDPHAT (45).
	Figure 5. Affinity of Asf1 and H3/H4 determined by fluorescence quenching. (A) Schematic diagram of model equilibria for yAsf1 association with H3/H4 complexes. D and T indicate H3/H4 dimers and tetramers, respectively. (B) H3/H4*Qsy9 binding to yAsf1* was observed by fluorescence quenching. H3/H4*Qsy9 was titrated into 1.0 nM yAsf1*. The data were fitted with a ligand-depleted binding model (Equation 1; GraphPad Prism) because the concentration of yAsf1* was within 10-fold of the Kd value. (C) H3/H4*Qsy9 binding to yAsf1*V94R was observed by fluorescence quenching. H3/H4*Qsy9 was titrated into 1.0 nM yAsf1*V94R. The curve was fitted (GraphPad Prism) with a Langmuir single-site binding isotherm.

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