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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 - J. Biol. Chem. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
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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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.
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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.

Mentions: We previously reported that Idas alone does not bind Cdt1 (24), whereas the affinity of an Idas-Geminin complex for Cdt1 is significantly reduced compared with Geminin based on surface plasmon resonance experiments. Here we used ITC to analyze the binding of the three dimers, tGeminin-tGeminin, tIdas-tGeminin, and tIdas-tIdas, to a truncated Cdt1 construct spanning residues 113–353 in solution and in more detail (Fig. 7). We measured that the affinity of tIdas-tGeminin for Cdt1 is about 35 nm, ∼10-fold lower than the affinity of the Geminin homodimer for Cdt1 (KD ∼ 3.5 nm).


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 - J. Biol. Chem. (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814758&req=5

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.
Mentions: We previously reported that Idas alone does not bind Cdt1 (24), whereas the affinity of an Idas-Geminin complex for Cdt1 is significantly reduced compared with Geminin based on surface plasmon resonance experiments. Here we used ITC to analyze the binding of the three dimers, tGeminin-tGeminin, tIdas-tGeminin, and tIdas-tIdas, to a truncated Cdt1 construct spanning residues 113–353 in solution and in more detail (Fig. 7). We measured that the affinity of tIdas-tGeminin for Cdt1 is about 35 nm, ∼10-fold lower than the affinity of the Geminin homodimer for Cdt1 (KD ∼ 3.5 nm).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus