HJURP^Scm3 is sufficient to assemble CENP-A nucleosomes in vitro. (A) Plasmid supercoiling assays were conducted using recombinant MBP-tagged HJURP^Scm3 and recombinant CENP-A octamer (including histones H4, H2A, and H2B) or histone H3.1 octamer to assess the relative ability of HJURP to assemble CENP-A– and H3.1-containing nucleosomes. The relaxed DNA lane contains topoisomerase-treated supercoiled (S.C.) plasmid DNA. HJURP^Scm3 induced supercoiling more efficiently in the presence of CENP-A relative to H3.1. (B) Line scans across topoisomers within conditions presented in A. Lines indicate the least supercoiled topoisomers. Boxes indicate the location of the maximally assembled topoisomers. (C) Assembly reactions from A containing H3.1 and CENP-A are graphed here as fold intensity over reactions containing no HJURP^Scm3. Error bars show standard deviations. (D) Assembly reactions in A (using HJURP^Scm3) or assembly reactions using NPM1 digested with micrococcal nuclease to show DNA protection of the assembled species. A dotted line was drawn to indicate the migration of a 200-bp fragment.