Fig. 3: An extended Cdt1 C-WHD is required to recruit half MCM onto DNA.
From: Stabilisation of half MCM ring by Cdt1 during DNA insertion
a Scheme illustrating the Cdt1 N terminus deletions used in the preRC assay. N terminus domain is shown in black (NTD), middle WHD in light grey (M-WHD) and C terminus WHD in dark grey (C-WHD). The long loop connecting both WHDs is shown in white (from amino acid 430–495). The first amino acid present in the Cdt1 truncations give the name to them. The S272 truncation contains a deletion of almost all NTD, while both domains, NTD and M-WHD, have been deleted in the S436 truncation. S460, M471 and A495 are different truncations affecting the length of the connecting loop and they are based on the OCCM structure (PDB: 5V8F). S460 truncation deletes mainly the loop section that interacts with Mcm2, while M471 keeps only the loop that interacts with Mcm6 C-WHD but not with the AAA+ domain. Finally, A495 deletes the extension of the helix 1 of Cdt1 C-WHD and abolishes any interaction with the Mcm6 C-WHD. b Recruitment of the MCM-Cdt1 by the OC complex (ATPγS and low salt washes), using Cdt1 wt (lane 1) or the different truncations (lanes 2–6). Negative control, where Cdt1 is omitted, is shown in lane 7. Under ATP hydrolysis, low salt wash is shown in c (release conditions) and high salt wash in d (loading conditions). e Loading of the MCM with increasing amounts of two different Cdt1 truncations (S460 and A495). The truncation that contains the smallest deletion (S272) is included as a positive control (Lane 1).
