Figure 2.

Rapid and efficient formation of mono-prenucleosomes with 80-bp DNA fragments. (A) The NAP1-mediated formation of mono-prenucleosomes with an 80-bp genomic DNA fragment occurs rapidly and requires all four core histones. Histone–NAP1 complexes were combined with an 80-bp DNA fragment (an 80-bp segment in the coding sequence of the Drosophila melanogaster ISWI gene; henceforth termed the “80-bp genomic DNA”). The samples were incubated at room temperature for 30 sec and then subjected to native (nondenaturing) 5% polyacrylamide gel electrophoresis. The DNA was visualized by staining with ethidium bromide. One octamer equivalent of all four core histones per DNA would be a 1:1 octamer:DNA ratio. Note that one octamer equivalent of H2A+H2B+H3+H4 has the same amount of H3 and H4 as one octamer equivalent of H3+H4. (B) Mono-prenucleosomes can be formed with the central 80 bp of the 601 nucleosome positioning sequence. Mono-prenucleosomes were formed and analyzed as in A, with all four core histones along with either the 80-bp genomic DNA or the central 80 bp of the 601 sequence. (C) Mono-prenucleosomes appear to be the thermodynamically most stable arrangement of the four core histones and 80 bp of DNA at 50 mM NaCl. Mono-prenucleosomes were formed with the dNLP histone chaperone as well as by salt dialysis of the four core histones with the 80-bp genomic DNA fragment. For comparison, H3–H4 monotetrasomes were also generated in parallel by salt dialysis with H3–H4. The histones were used at an octamer equivalent:DNA ratio of 1.0. (D) Mono-tetrasomes can be converted into prenucleosomes by the addition of H2A–H2B. Monotetrasomes were formed by salt dialysis with the 80 bp of genomic DNA as in C. Next, NAP1–H2A–H2B complexes were added as indicated. The samples were incubated for 30 sec at room temperature and then subjected to native 5% polyacrylamide gel electrophoresis. As a reference, a mono-prenucleosome formed by salt dialysis as in C was included (“Mono-prenuc”).