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. 2022 Apr 14;29(4):403–413. doi: 10.1038/s41594-022-00758-y

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 5 — a) FRET analysis of homotypic CENP-A mono-nucleosome (CA) or H3 mono-nucleosome (H3) interactions in the absence or presence of CENP-N. Donor is a mono-nucleosomes containing Alexa 488 labeled H2B; Acceptor is a mono-nucleosome containing Atto N 647 labeled H2B. 250 nM donor and acceptor nucleosome; FRET intensity in dependence of [CENP-N]. Final concentration for NaCl is around 100 mM. Error bars from three independent measurements. Data are presented as mean values + /- SD. b) Salt concentration affects nucleosome stack formation. AUC analysis (van Holde-Weischet plots) of CENP-N in complex with CENP-A mono-nucleosomes at 60 and 200 mM NaCl). CN: CENP-N^1–289 in complex with CENP-A mono-nucleosome. CA_MN: CENP-A mono-nucleosome. c) CENP-N mutant (K102A) binds to CENP-A nucleosomes as well as wild-type CENP-N (5% native PAGE). 250 nM CENP-A nucleosome was combined with CENP-N at ratios of 2:1, 4:1 and 8:1 in buffer containing 50 mM NaCl, 20 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM DTT. d) deletion of the H4 N-terminal tail (∆19) does not affect the specific interaction between CENP-N and CENP-A nucleosomes. CENP-N was mixed with CENP-A nucleosome containing full length H4 or (∆19) H4 at a 2:1 ratio in the same buffer as in A).

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