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. 2021 Jun 23;12:3883. doi: 10.1038/s41467-021-24199-1

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© The Author(s) 2021

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. 3 — a DNA binding β-hairpin structure in the Cdc6 WHD. The four lysine residues contacting DNA are shown as sticks. b The new DNA binding Arg-267 in the Orc6-BP in the ORC–Cdc6 structure. c The first and fourth DNA-interacting Cdc6 β-hairpin lysine residues are conserved in eukaryotes. d Pre-RC reaction with Cdc6 wild-type (WT), Cdc6 K477A/K483A (2A-1), Cdc6 K479A/K481A (2A-2), or Cdc6 K477A/K479A/K481A/K483A (4A or 4Ala) was assembled, washed with high salt, and analyzed by silver staining. Purified Orc1 (104 kDa), Mcm5 (86 kDa), Cdt1 (68 kDa), and Cdc6 (58 kDa) are used as molecular weight markers. e Mcm5 intensities of the Mcm2-7 double hexamer in (d) were quantified by ImageJ (version 1.50i) and the relative Mcm5 intensities were plotted using three independent experiments. The center for the error bar is the average of three measurements. The amount of Mcm5 was used as an indicator for the Mcm2-7 loading efficiency of the WT ORC in partner with the mutant Cdc6. Each reaction was done three times independently. Individual data points are shown as gray dots. Error bars represent standard deviations.