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. 2000 Jun 6;97(13):7266–7271. doi: 10.1073/pnas.130189697

Figure 1.

Figure 1

Topo I analyses using the purified CENP-A, histone H4, H2A, and H2B. (A) Samples from each step of purification were identified by silver staining (lanes 1–4) or by immunoblotting with anti-centromere auto-antibody serum (lane 5) after 13% SDS/PAGE. A crude HCl extract of HeLa nuclei (lane 1), peak fraction of CENP-A after first (lane 2) and second (lane 3) reversed-phase HPLC, and CENP-A purified by SDS/PAGE and electro-elution (lanes 4 and 5) are shown. CENP-A monomer (17 kDa) and dimer (34 kDa) are indicated. (B) H3/H4 (lanes 1–6), CENP-A/H4 (lanes 7–12), or H4 (lanes 13–18) were subjected to Topo I analyses using the relaxed form of closed circular DNA (pUC-αdimer, 20 ng) with (lanes 1–3, 7–9, and 13–15) or without NAP-1 (lanes 4–6, 10–12, and 16–18). Supercoil ladders were detected by Southern hybridization. H3/H4: 30 ng (lanes 1 and 4), 50 ng (lanes 2 and 5), 75 ng (lanes 3 and 6); CENP-A/H4: 10 ng (lanes 7 and 10), 20 ng (lanes 8 and 11), 30 ng (lanes 9 and 12); H4: 25 ng (lanes 13 and 16), 50 ng (lanes 14 and 17), and 75 ng (lanes 15 and 18). (C) Reconstituted core histones (H3/H4/H2A/H2B) (lanes 1–6), CENP-A/core histones (CENP-A/H4/H2A/H2B) (lanes 7–10), or H3(-) core histones (H4/H2A/H2B) (lanes 11–14) were subjected to Topo I analyses with (lanes 1–3, 7, 8, 11, and 12) or without NAP-1 (lanes 4–6, 9, 10, 13, and 14) under the same conditions as in B. H3/H4/H2A/H2B: 25 ng (lanes 1 and 4), 35 ng (lanes 2 and 5), 50 ng (lanes 3 and 6); CENP-A/H4/H2A/H2B: 40 ng (lanes 7 and 9), 60 ng (lanes 8 and 10); H4/H2A/H2B: 40 ng (lanes 11 and 13), and 60 ng (lanes 12 and 14).