Fig. 2: Investigating the oligomerisation state of the CDT1-geminin complex.
From: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions
a Substitution of CDT1 with a co-expressed CDT1-geminin complex (+CG, −CDT1ΔN, lanes 3 and 6) inhibits pre-RC formation to a comparable extent as adding geminin (GemFL, lanes 2 and 5). *red = CDC6, *purple = ORC6, *green = geminin, *blue = CDT1. CDT1 runs as a doublet due to proteolytic degradation. Representative of three biological repeats. b Titration of geminin into the pre-RC assay, showing that at least a 2× excess (1:2 CDT1ΔN:GemFL = CDT1 monomer to a geminin dimer) is required to inhibit DNA licensing. c Quantification of MCM2-7 loading from (b). No significant change in inhibition is observed when adding >2× geminin. Shown as the mean ± SD of three biological repeats, normalised to the pre-RC control and analysed using one-way ANOVA with Dunnett’s multiple comparisons test. **p = from bottom to top; 0.0011, 0.0017, 0.0032. d Mass photometry measurements of CDT1ΔN (blue), geminin (GemFL, green) and CDT1 + geminin (CDT1ΔN + GemFL, yellow) showing that CDT1ΔN and geminin form a complex of ~90 kDa, that is consistent with the hetero-trimer (Tri., indicated by the grey shading). e Mass photometry exploring previously reported CDT1:geminin ratios (1:2 = hetero-trimer (yellow), 1:3 = hetero-tetramer (green), 1:4 = hetero-pentamer (blue), 2:4 = hetero-hexamer (red)). An excess of geminin results in reduced relative counts of the CDT1-geminin complex due to saturation of the movie frames with uncomplexed geminin dimers. The grey shading indicates the expected masses of each oligomeric state (abbreviated as Tri, Tet., Pent. and Hex.). f Expected molecular weights (MW) of CDT1ΔN, geminin and oligomeric complexes at the ratios detailed. Observed MW obtained from mass photometry measurements in parts (d, e) and from SEC-MALS of the CG complex from part (a). Geminin forms a dimer in solution (MW 48 kDa), and a hetero-trimer (1:2 CDT1ΔN:geminin) is observed by mass photometry and SEC-MALS under all ratios tested. Data representative of two biological repeats and shown as the mean ± SE. Source data are provided as a Source Data file.
