HJURP^Scm3-assembled CENP-A nucleosomes are negatively supercoiled and contain H2A and H2B. (A and B) Supercoiling assay comparing assembly efficiencies of chaperones dNAP, NPM1, and HJURP^Scm3 with CENP-A histone octamers (CENP-A–H4 and H2A/H2B) in A or with CENP-A–H4 alone in B. CENP-A–H4 levels added to the reactions were varied from 1 to 2× compared with the amount of CENP-A–H4 present in the reactions in A. Line scans are presented in Fig. S3. SC, supercoiled. (C) Integrated intensities of maximally supercoiled populations were measured from reactions in A and B. Values are graphed as fold-maximally supercoiled heterotetramer to octamer. Error bars show standard deviations. (D) Supercoiling assay showing assembly activities (top) for dNAP, HJURP^Scm3, and NPM1. Supercoiled DNA was separated by agarose gel electrophoresis with (bottom) or without (top) the DNA intercalating agent chloroquine to distinguish negatively and positively supercoiled DNA. The minus signs indicate no addition of chaperone. The white line indicates that intervening lanes have been spliced out.