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. 2018 Mar 15;29(6):751–762. doi: 10.1091/mbc.E17-10-0596

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© 2018 Cao, Zhou, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 1: — CENP-N protects CENP-A nucleosomes from micrococcal nuclease digestion. (A) Comparative MNase digestion analysis of H3.1- and CENP-A nucleosomes, reconstituted with a 166–base pair DNA fragment derived from the 601 sequence. After digestion (quenched with 50 mM EDTA), samples were analyzed in a gel shift assay prior to DNA extraction (left panel) and after DNA extraction (right panel). The length of the DNA fragments was determined according to the standard DNA ladder curve obtained from the Bioanalyzer (Agilent). (B) MNase digestion analysis of CENP-A nucleosome (with the same DNA sequence as in A) in the absence and presence of CENP-N^1-289. The reactions were performed and analyzed as in A. (C) MNase digestion analysis of CENP-A nucleosome (147–base pair DNA derived from α satellite DNA) in the absence and presence of CENP-N^1-289. (D) The DNA ends of the CENP-A nucleosome are important for the proper orientation of CENP-N^1-289 on the CENP-A nucleosome. CENP-A nucleosomes were reconstituted with either 147 or 127 base pairs of 601 nucleosome-positioning DNA. CENP-N^1-289 was mixed with CENP-A nucleosome at a 3:1 ratio. After a 5-min incubation at 37°C, samples were analyzed by 5% native PAGE.