Fig. 5: Interaction with the Orc4 loop by Mcm6 C-WHD is not essential for Cdt1 stabilization of half MCM.
From: Stabilisation of half MCM ring by Cdt1 during DNA insertion
a Cryo-EM structure of the OCCM complex8. The ORC-Cdc6 ring is illustrated in light grey mesh, while the MCM-Cdt1 ring is in dark grey mesh. The surface of specific individual proteins are highlighted in different colours: Orc 4 (green), Orc5 (blue), Mcm6 (black), and Cdt1 (orange). Of these, the domains studied in this work are further highlighted with their secondary structure, maintaining the colours (extended C-WHD of Cdt1, C-WHD of Mcm6, Orc4 C-WHD and Orc5 AAA lid). Note that the secondary structure of Mcm3 C-WHD is also illustrated in grey. The zoomed area shows in more detail the interaction between the Orc4 loop (shown in green) and the C-WHDs of Mcm6 and Cdt1 (shown in black and orange, respectively). Three different amino acid substitutions, in the C-WHDs of Mcm6 and Cdt1, affecting the interaction with the Orc4 loop were studied. First, contacts at the beginning and the end of the Orc4 loop by the C-WHD of Mcm6 were mutated (Mcm6 2-1, shown in magenta in the zoomed section). Second, different residues present in the main helix of Mcm6 that establish contacts with Orc4 loop were substituted with different amino acids going from mild to severe changes (Mcm6 2-2, 2-3 and 2-4, shown in cyan. See also Supplementary Fig. 5). Third, the contacts between the Orc4 loop and the C-WHD of Cdt1 were mutated in Cdt1 2-1 (in orange). Specific substitutions present in the different proteins mutated are summarized in the box. b Recruitment of MCM-Cdt1, containing Mcm6 wt, 2-1, 2-2, 2-3 and 2-4 mutations (lanes 1-5). Mcm6 1-1 is included as negative control in lane 6. c Release and loading of the complexes analysed in b. The recruitment and loading of the MCM-Cdt1 complex containing Cdt1 2-1 mutation is shown in Supplementary Fig. 6a, b.
