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. 2020 Feb 10;11:819. doi: 10.1038/s41467-020-14563-y

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© The Author(s) 2020

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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Fig. 4 — a In vitro pull-down (PD) assay using purified components. 6xHis-MBP-tagged PALB2 ChAM domain or 6xHis-MBP immobilised on Ni-NTA affinity beads, incubated with recombinant nucleosome variants. Acidic patch mutant (Ac-patch mut) comprises H2A E61/91/92A and H2B E105A; ΔH3 NT comprises H3 residues 25−135; ΔH4 NT comprises H4 residues 21−106. Anti-H3 and H4 antibodies showed increased reactivity towards histones with shorter N-terminal tails. b The nucleosome and its acidic patch. Left panel: electrostatic potential view of the protein histone surface; right panel: ribbon representation of the nucleosome core particle. c Sequence alignment of ChAM with alanine scanning mutation areas highlighted (blue line) and basic residues mutated in 4 M mutant highlighted (red arrows). d, e Clonogenic survivals of RPE1 p53 KO (e) or p53/BRCA1/53BP1 KO (f) cells complemented with PALB2^WT, PALB2^∆ChAM or PALB2^4M in response to indicated doses of olaparib. Lower panels show corresponding area under curve (AUC) bar graphs; n ≥ 3. The bars represent mean ± s.e.m.; one-way ANOVA; **P < 0.01, ***P < 0.001; ns, not significant (P ≥ 0.05); n = 3 independent experiments. Source data are provided as a Source Data file. f Pull-down assays comparing NCP association of 6xHis-MBP-PALB2 ChAM variants with charge removal and charge retaining ChAM mutations on Arg414 and Arg421. g Pull-down assays between NCPs and immobilised 6xHis-MBP-ChAM variants containing alanine mutations across the proposed nucleosome-interacting region.