Caillat C et al. (2013), The Geminin and Idas coiled coils preferentiall...

XB-ART-47878

J Biol Chem 2013 Nov 01;28844:31624-34. doi: 10.1074/jbc.M113.491928.

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The Geminin and Idas coiled coils preferentially form a heterodimer that inhibits Geminin function in DNA replication licensing.

Caillat C , Pefani DE , Gillespie PJ , Taraviras S , Blow JJ , Lygerou Z , Perrakis A .

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Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.

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Species referenced: Xenopus laevis
Genes referenced: cdt1 gmnn lhx6 mcidas

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	FIGURE 1. Idas and Geminin form a stoichiometric complex after co-expression of Geminin and Idas in bacteria and purification by using immobilized metal affinity chromatography (IMAC). The SDS gel shows the protein marker (M), total cell extract (T), soluble fraction (S), elution (E), and elution fraction after cleavage of the his tag (C).
	FIGURE 2. SAXS analysis of Geminin, Idas, and the Idas-Geminin complex. A, the scattering curves for tGeminin, tIdas, and tIdas-tGeminin complexes; only the low angles are shown for clarity. B, Guinier plots and distance probability distribution functions for the same complexes.
	FIGURE 3. Stability analysis of the different Idas and Geminin dimers. A, the ratio of fluorescence intensities at 350 and 330 nm (indicative of folding) as a function of temperature, used to calculate the Tm valueσ. B, the Tm values derived from the experimental data. C, a pulldown assay on the purified homodimers shows that the association of the tGeminin dimers is irreversible. Purified his-Geminin:tGemnin was mixed with purified tIdas-tIdas (S) and immobilized on Talon beads. The different fraction are: flow-through (FT), wash (W), elution (E).
	FIGURE 4. Idas is the preferred dimerization partner for Geminin. U2OS cells were (co)transfected with full-length Geminin-GFP, Geminin-HA, and/Idas-GFP or GFP as indicated. Cells transfected with GemininGFP alone served as the negative control. Total cell lysates (left) and anti-HA immunoprecipitates (IP, right) were immunoblotted for GFP (upper) and HA (lower). When both Idas and Geminin were available in similar amounts, Geminin preferred to heterodimerize with Idas.
	FIGURE 5. The structure of the Idas-Geminin complex. A, a diagram of the domain structure for Geminin and Idas·coiled-coil domain (CC), destruction box (DB), homeodomain binding region (HD). The limits of the crystallization construct (tIdas-tGeminin) and those of the coiled coil are numbered and denoted as thicker boxes. B, a sequence alignment of the seven heptads involved in the coiled coil. C, a schematic representation of the crystal structure of the tGeminin-tIdas structure. Differences in the a and d register positions are in bold. D–F, the structures of the Geminin homodimer, alone (D) and from the complex with Cdt1 (E and F).
	FIGURE 6. Interactions in the coiled coil dimerization interface. Shown are the interactions in the a1, a5, and d2 positions in the tIdas-tGeminin complex (A, C, and E) and in the tGeminin-tGeminin complex (B, D, and F). G, a lookup table for all non-hydrophobic interactions of coiled coil region for the tIdas-tGeminin complex (left half) and the tGeminin-tGeminin complex (right half). The residue names and numbers, coiled coil register positions, and atom names are indicated.
	FIGURE 7. The tIdas-tGeminin complex binds Cdt1 with less affinity and different thermodynamic characteristics than the tGeminin-tGeminin. A and B, isothermal titration calorimetry data recorded upon successive injections of tGeminin-tGeminin or tGeminin-tIdas into a cell containing tCdt1 and the analysis. The derived values for the KD and the change in free energy (ΔG), enthalpy (ΔH), and entropy (−ΤΔS) are shown; the small difference in the change of the free energy of binding in fact results from large changes in the relative contribution of enthalpy and entropy.
	FIGURE 8. Idas inhibits Geminin function as an inhibitor of DNA replication in Xenopus egg extracts. A, increasing concentrations of tIdas-tIdas, tIdas-tGeminin, and tGeminin-tGeminin were assessed for their ability to inhibit DNA replication. DNA replication is presented as a percentage relative to 100% replication of buffer treated extract. S.E. are derived from three independent experiments. B, the same for the dIdas and dGeminin constructs.
	FIGURE 9. Overexpression of Idas in U2OS cells results in increased DNA content. A, full-length Idas-GFP, Idas-GFP and Geminin-HA, or GFP were transfected in U2OS cells. 52 h post transfection cells were fixed, and DNA was stained with Hoechst. DNA content in each transfected cell was quantified by measuring the total intensity of Hoechst staining per nucleus using Image J. The results are presented both as a histogram (bars showing the percentage of cells within a range of arbitrary intensity units) and as a cumulative distribution (lines showing the percentage of cells below a certain intensity). Idas-GFP positive cells show an increased number of cells with high Hoechst intensity compared with the GFP control; co-expression of Geminin decreases the number of cells with high DNA content both compared with Idas and to the GFP control. B, U2OS cells transfected with Idas-GFP and GFP were stained for γH2AX, which marks double-strand breaks. Idas-GFP transfected cells present an increased percentage of cells staining positive for γH2AX compared with cells transfected by GFP alone, indicating over-replication. Error bars are derived from three independent experiments.

References [+] :

Acharya, Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R). 2006, Pubmed