Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

Abstract

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

Fig. 1.

Structure of the human tCdt1:tGeminin complex. The (2x[Cdt1:2xGeminin]) heterohexamer is shown as a cartoon representation: Cdt1 molecules in orange, Geminin molecules in green and blue shades. The primary, secondary, and the novel tertiary interface regions are boxed, in a magenta-, pink-, and blue-colored box, respectively. We also show a schematic representation of the Cdt1 and Geminin proteins; the 3 interfaces are marked in the sequence and shown with the same color scheme (additional functional regions are shown with labeled bars).

Fig. 2.

The tripartite human htCdt1:htGeminin interface. The primary (A), secondary (B), and tertiary (C) interfaces are shown (coloring and bounding boxes as in Fig. 1). Interacting residues are shown as a stick model; carbon atoms follow the cartoon coloring; oxygens, nitrogens, and sulfurs are in red, blue, and green. Important residues are labeled; mutation sites in Cdt1 that are used in this study have labels underlined (the residues deleted in hvtGem are grayed out). (D) A multiple-sequence alignment of the sequence regions involved in the formation of the novel tertiary interface; residues in orange for Cdt1 and dark green for Geminin are the ones depicted also in (C). Arrowheads denote the mutation sites on the Cdt1 tertiary interface, the vertical bar and the green shading box indicate the Geminin deletion construct, and the dark yellow bounding box the region substituted in the grafting mutations (residues 139–150).

Fig. 3.

Solution analysis of htCdt1:htGeminin and htCdt1:hvtGeminin by SAXS. (A) The experimental SAXS profile (log intensity as a function of the momentum transfer) of htCdt1:htGem (gray curve) is compared with the theoretical scattering curves calculated from the corresponding crystallographic models of the complex as a heterohexamer (blue line and cartoon) and a heterotrimer (magenta line and cartoon), respectively. (B) The ab initio calculated SAXS model of htCdt1:htGeminin (depicted as light gray spheres) is superimposed to the crystal structure of htCdt1:htGeminin heterohexamer (Cα trace colored as in Fig. 1). (B and D) The same as (A) and (B) but for htCdt1:hvtGeminin.

Fig. 4.

Correlation between Geminin mutants activity and quaternary arrangement of the Cdt1:Geminin complex. (A) htGem, hvtGem, htGem-GCN4α, and htGem-GCN4β were tested for their ability to inhibit DNA replication in Xenopus egg extracts. For each construct, 6 concentrations were tested: 480, 240, 120, 60, 30, and 15 nM; a Coomassie blue-stained gel was used for loading control and is shown. For the graph, 100% replication was adjusted based on buffer-treated extracts, and error bars represent standard errors of the mean. (B) The experimental SAXS profile of htCdt1:htGem-GCN4α (Left) and htCdt1:htGem-GCN4β (Right) are compared with the theoretical scattering curves calculated from the heterohexamer and heterotrimer. Only the low-angle regions are shown for clarity; the coloring scheme is as in Fig. 3 A and D.

Fig. 5.

Functional significance of tertiary interface Cdt1 mutants. (A) WT human Cdt1 fragment 158–546 and 2 mutants (R210A and R198A/R210A) were tested for their ability to rescue DNA replication in Cdt1-depleted Xenopus egg extracts. For each construct, four concentrations were tested: 30, 15, 7, and 3.5 nM. One hundred percent represents replication in non–immune-depleted extracts. Error bars represent standard errors of the mean. Equal loading of Cdt1 constructs was verified by Coomassie staining and Western blot analysis with anti-His antibody. (B) Rereplication in U2OS cells with reduced cyclin A activity is different when wtCdt1 or the 2 interface mutants (R210A, R198A/R210) are overexpressed. The panel reports the mean and the standard error of the mean of 4 independent experiments; the baseline 10%, representing the mean amount of cells with rereplicated DNA in cells with reduced cyclin A.

Fig. 6.

Proposed model for the mechanism of DNA licensing inhibition by Geminin.