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. 2010 Feb 18;285(16):11913–11921. doi: 10.1074/jbc.M109.083238

FIGURE 3.

FIGURE 3.

Topological assay for nucleosome assembly by sNASP. A, relaxed ϕX174 DNA (10 ng/μl), which was previously treated with wheat germ topoisomerase I (lane 2), was incubated with hNap1 (lanes 4–6) or sNASP (lanes 9–11) in the presence of core histones. The reaction products were then analyzed by 1% agarose gel electrophoresis in 1 × TAE buffer. Lanes 3 and 8 indicate negative control experiments without histone chaperone in the presence of core histones. Lanes 7 and 12 indicate the other negative control experiments without core histones in the presence of hNap1 (1 μm) or sNASP (1 μm). The concentrations of hNap1 and sNASP were 0.25 μm (lanes 4 and 9), 0.5 μm (lanes 5 and 10), and 1 μm (lanes 6 and 11). B, tetrasome assembly is shown. Reactions were conducted as described for the experiments shown in panel A, except H2A·H2B or H3.1·H4 were used instead of the four core histones, H2A·H2B/H3.1·H4. Lanes 3 and 8 indicate negative control experiments without histone chaperone in the presence of H2A·H2B or H3.1·H4, respectively. Lanes 5, 7, 10, and 12 indicate the other negative control experiments without core histones in the presence of either hNap1 (1 μm) or sNASP (1 μm). Lanes 3, 4, and 6 represent experiments with H2A·H2B, and lanes 8, 9, and 11 represent experiments with H3.1·H4.