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.