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. 2015 May 15;6(2):252–266. doi: 10.3390/genes6020252

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© 2015 by the authors; licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).

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Defective Geminin mutants bind Cdt1. (A) Geminin missense mutants bind Cdt1. Recombinant Xenopus Geminin proteins were mixed with translated Myc-Cdt1. The reactions were precipitated with anti-Myc antibody, and the precipitate was blotted with the indicated antibodies. (B) Inhibition of DNA replication by Geminin mutants. Recombinant Geminin proteins were added to replication extracts made from Xenopus eggs at the indicated concentrations, and the extent of sperm DNA replication was measured. Average of two experiments. (C) Geminin^AWA competes away Geminin^WT. Recombinant Geminin^WT (2 ng/µL) and an excess of a Geminin mutant (50 ng/µL) were added to replication extracts, and the extent of DNA replication was measured. Average of two experiments. (D) Geminin^AWA and Geminin^WT have a similar binding affinity for Cdt1. The binding affinity between recombinant mouse Cdt1^172–368 and either WT or AWA mouse Geminin^72–157 was measured by isothermal calorimetry.