A) Nucleosomes were assembled with yeast histones and 181 bp DNA fragments with variants of the sea urchin 5S rDNA NPS in which the native DraI site was moved from position 78 to the sites indicated (). The NPS identified in animal histones () is shown in the grey bar, and the observed location of yeast histone cores on these DNA fragments is shown in blue. B) Results of a typical DraI digestion with a 181 bp DraI-78 nucleosome with or without yFACT. C) DraI digestion rates were calculated for nucleosomes alone, with Nhp6 added, or with yFACT added (Nhp6 and Spt16-Pob3). Averages and standard deviations from at least three measurements with each variant template are shown (). Results for DraI-152, DraI-157, and free DNA are given above the chart and in or . D) As in panel A, except templates with a recognition site for PstI are shown with the observed position of histone cores in green. E) The approximate locations of the recognition sites for DraI and PstI are mapped onto the structure of the yeast nucleosome () assuming alignment of the left ends of the linear DNAs in each case. DNA regions contacted by H2A–H2B are colored red, and those contacted by H3–H4 are colored blue. An intact nucleosome is shown on the left, with just the DNA shown for the variant template sets. Red (DraI) or green (PstI) spheres indicate the phosphodiester linkage digested. F) As in panel C, except the rates of PstI digestion are shown for the templates in panel D.

A 146 bp 5S rDNA fragment () labeled with ³²P at the 5' end of the bottom strand (*) was used for hydroxyl radical analysis (Experimental Procedures). The trace here shows the region below the intact 146 nt band. Each scan is from the same gel and is presented on the same scale, so the height of individual peaks represents the relative yield of that product under identical conditions. Grey boxes on the cartoon indicate the undigested 146 nt band, the region 30 bp from the left end of the DNA fragment (with arrows on the nucleosomal traces), the center of the DNA fragment/nucleosomal dyad, and the last product resolved on the gel (~25 nt).

A) Nucleosomes (181 bp 5S rDNA, yeast histones) were incubated for 10 minutes at 30° under NaCl-HSA conditions with or without 10 mM MgCl₂ (Experimental Procedures) and with or without yFACT. Aliquots were then subjected to native PAGE either without (lanes 1–4) or with (lanes 5–8) addition of excess unlabeled genomic DNA. The single gel with 8 lanes was scanned to detect DNA (Cy5, labeled "a" in blue) and H2A (Oregon Green 488, labeled "b" in green), then the panels were reassembled to simplify comparison. The regions containing free DNA (DNA), nucleosomes (Nuc), and intact yFACT-nucleosome complexes (yFACT-Nuc) are indicated. The asterisk indicates the position of Spt16-Pob3 complexes with H2A–H2B dimers, and the red arrow indicates segment 38, the last one included in calculation of the material in the displaced region (Displaced). The segment numbering scale for quantitation is shown on the right. B–D) The region from the well to the free DNA in lanes 5 and 7 was quantitated in 100 segments, then the signals were normalized by subtracting the background level and dividing by the total amount of signal for lane 5. Panels B–D show the same data adjusted emphasize different features.

A) A nucleosome (181 bp 5S rDNA, yeast histones) with (lane 1) or without (lane 2) yFACT, and a similar amount of purified yeast H2A–H2B dimer (lane 3) were incubated under NaCl-HSA conditions without MgCl₂ (Experimental Procedures) at 30° for 10 minutes. Unlabeled genomic DNA was added to all samples prior to native PAGE analysis as in . Quantitation of segments 1–52 is shown in panel B. Curves are labeled with the lanes scanned, and segment 38 is indicated with a red arrow. C) Three repeats of the nucleosome samples in panel A were analyzed, except MgCl₂ was added as indicated and the incubation was at 37°. The amount of signal in segments 1–38 was determined with the average and standard deviation plotted. The difference between the H2A and DNA values was calculated (shown in the row labeled "H2A – DNA"), then the difference between these values for the samples with and without yFACT was determined (shown in the "+/− yFACT" row) to give the amount of dimer displaced as a result of yFACT binding. D) An experiment similar to the one in panel C was performed, except the nucleosome was assembled with X. laevis histones, H4 was labeled in addition to the H2B and DNA, and samples were incubated at 30°. Samples were then analyzed as in panel C.

Nucleosomes were assembled with yeast histones and the 181 bp 5S rDNA DraI-78 fragment and digested with DraI with or without yFACT, or incubated under the same conditions without DraI for analysis by native PAGE after adding excess unlabeled genomic DNA. The top panel shows the dimer and DNA distributions for each set of samples after native PAGE, the middle panel shows the accumulation of digestion products with time, and the bottom panel shows analysis of the "displaced" region of the native gel for the 32 minute time points, as in . The total dimer loss due to yFACT binding as calculated in was 20%.

W303 strains were constructed with the GAL1-10 promoter integrated adjacent to the 7887 bp YLR454w ORF (), both copies of the genes encoding H2A and H2B deleted, and a plasmid expressing H2A and an N-terminal fusion of the FLAG epitope to H2B (generously provided by MA Osley; ). Versions with the POB3 (WT; DY13736) or pob3-L78R alleles (DY13744) were grown at 25° in raffinose medium, then the promoter was induced by the addition of 2% galactose. Samples were collected at times after induction as indicated, then H2B and H3 occupancy was measured by ChIP and quantitative PCR using primers for the promoter (−192 to +168 relative to the ORF), the 5' end (+945 to +1147), and the 3' end (+7701 to +7850) (Experimental Procedures). PCR was performed in triplicate with each primer set, with the standard deviation shown (some error bars are obscured by the data symbol).

See text for details. Reinsertion of dimers from a free pool may be mediated by yFACT or by other factors.