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. 2011 May 17;2:313. doi: 10.1038/ncomms1320

Figure 4. Cse4-nucleosomes do not preferentially assemble on CEN3 DNA.

Figure 4

(a) Experimental scheme of Cse4- and Sc-nucleosomes reconstitution on 5S, 601 and CEN3 DNA. Nucleosomes were reconstituted under limiting octamer concentration by salt dilution on either a 207-bp atto647-labelled CEN3 or cy3-labelled 601 DNA probe in the presence of an excess of 147-bp 5S DNA and analysed by 5% native PAGE. (b) The reconstitution on total DNA (≈5S DNA) was visualized by ethidium bromide staining. Nucleosomes assembled on 601 DNA and CEN3 DNA were detected by scanning for cy3 601 (c) and atto647 (d), respectively. The ratio of octamer to DNA and the types of histone octamer (Cse4, Sc) used are indicated above the lanes. (e) Quantification of nucleosome assembly. The fraction of DNA incorporated into nucleosomes in b–d was calculated by dividing the signal of the shifted band(s) by the sum of free DNA and shifted band signal, and multiplying by 100. We excluded the material in the wells, but added all nucleosome bands. The graph of nucleosomes reconstituted using octamer–DNA ratio of 0.32:1 (Cse4-Nuc, shown in white) and 0.28:1 (Sc-nuc, shown in black) is shown. The mean and standard deviation values from 4 to 5 different experiments were used to plot the graph.