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. 2009 May 4;185(3):397–407. doi: 10.1083/jcb.200903088

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© 2009 Perpelescu et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — RSF complex can reconstitute and regularly space CENP-A nucleosomes using CENP-A core histones and naked DNA template. (A) Partially purified Flag-tagged (2 µl, left) and native (35 µl, right) RSF fractions were resolved on 7.5% SDS-PAGE and visualized with Coomassie Brilliant blue. Concentration of Rsf-1 was measured by densitometer tracing as 50 ng/µl for recombinant and 1 ng/µl for native fraction using BSA as control protein. (B) 15% SDS-PAGE stained with Coomassie Brilliant blue showing an equimolar ratio of reconstituted H3 (left) and CENP-A (right) core histones. CENP-A = ∼17 kD. (C) RSF-mediated H3 and CENP-A nucleosomes assembly assay. The ability of the RSF complex to reconstitute and space H3 and CENP-A nucleosomes was evaluated by the presence of periodic nucleosomal ladders following partial micrococcal nuclease digestion at two different dilutions (lanes 1–14) or a single dilution (15–18). Reactions were performed without (lanes 1–2), or with 10 µl each of recombinant (lanes 3–8) or native RSF fraction (9–18). The native fraction's activity was inhibited by immunodepletion with anti-Rsf-1 antibody (lane 17) and restored by the readdition of the same RSF sample (lane 18). Immunodepletion reduced Rsf-1 to ∼15% as detected from the analysis of the immunoblot shown under lanes 16 and 17. The nucleosomal ladder was resolved on a 1% agarose gel and stained with ethidium bromide.