Journal: Nature Communications
Article Title: Geminin inhibits DNA replication licensing by sterically blocking CDT1-MCM2 interactions
doi: 10.1038/s41467-025-67073-0
Figure Lengend Snippet: a Substitution of CDT1 with a co-expressed CDT1-geminin complex (+CG, −CDT1 ΔN , lanes 3 and 6) inhibits pre-RC formation to a comparable extent as adding geminin (Gem FL , 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 CDT1 ΔN :Gem FL = 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 CDT1 ΔN (blue), geminin (Gem FL , green) and CDT1 + geminin (CDT1 ΔN + Gem FL , yellow) showing that CDT1 ΔN 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 (M W ) of CDT1 ΔN , geminin and oligomeric complexes at the ratios detailed. Observed M W 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 (M W 48 kDa), and a hetero-trimer (1:2 CDT1 ΔN :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 file.
Article Snippet: This apparatus was coupled with a miniDAWN TREOS MALS unit and an Optilab T-rEX dRI detector (Wyatt Technology).
Techniques: Titration, Inhibition, Control
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