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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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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.
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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.

Mentions: Having established that Idas-Geminin interacts with Cdt1, albeit with lower affinity compared with Geminin-Geminin, we next wanted to study the biochemical function of the different complexes, Geminin-Geminin, Idas-Geminin, and Idas-Idas, in their ability to inhibit replication licensing and subsequent replication of sperm DNA in a Xenopus egg extract (Fig. 8). The assay in Xenopus egg extract allows measuring the activity of the purified homo or heterodimers in a system that supports DNA replication under the same control mechanisms as in early embryonic cells (41). First we used the coiled-coil domain alone (Fig. 8A), tGeminin and tIdas, as it has been shown that this truncated Geminin construct is competent for efficiently inhibiting licensing (21). tGeminin showed efficient inhibitory activity on DNA replication, with an IC50 of ∼50 nm, consistent with previous studies (20). The tIdas-tGeminin heterodimer inhibited DNA replication less efficiently, with an IC50 of ∼720 nm. Finally, the tIdas homodimer had no effect on DNA replication. We confirmed these results using the longer constructs of Geminin and Idas, dGeminin and dIdas. These constructs have similar inhibitory activities as the shorter forms (Fig. 8B). These findings correlate well with the lower affinity of tIdas-tGeminin for Cdt1 that we observed in vitro.


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)

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

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

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.
Mentions: Having established that Idas-Geminin interacts with Cdt1, albeit with lower affinity compared with Geminin-Geminin, we next wanted to study the biochemical function of the different complexes, Geminin-Geminin, Idas-Geminin, and Idas-Idas, in their ability to inhibit replication licensing and subsequent replication of sperm DNA in a Xenopus egg extract (Fig. 8). The assay in Xenopus egg extract allows measuring the activity of the purified homo or heterodimers in a system that supports DNA replication under the same control mechanisms as in early embryonic cells (41). First we used the coiled-coil domain alone (Fig. 8A), tGeminin and tIdas, as it has been shown that this truncated Geminin construct is competent for efficiently inhibiting licensing (21). tGeminin showed efficient inhibitory activity on DNA replication, with an IC50 of ∼50 nm, consistent with previous studies (20). The tIdas-tGeminin heterodimer inhibited DNA replication less efficiently, with an IC50 of ∼720 nm. Finally, the tIdas homodimer had no effect on DNA replication. We confirmed these results using the longer constructs of Geminin and Idas, dGeminin and dIdas. These constructs have similar inhibitory activities as the shorter forms (Fig. 8B). These findings correlate well with the lower affinity of tIdas-tGeminin for Cdt1 that we observed in vitro.

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