An inactive geminin mutant that binds cdt1

Abstract

The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7), the replication helicase, into the pre-replication complex (pre-RC) at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

Keywords: Cdt1; DNA replication; HBO1; geminin; pre-replication complex.

Figure 1

Structure and sequence of Geminin. (Top) Diagrammatic representation of a Geminin monomer. Black bars, Cdt1 binding sites; D-box, destruction box required in cis for ubiquitylation; NLS, nuclear localization signal. (Bottom) Sequence alignment of Human, mouse and Xenopus Geminin. Conserved residues are shaded. Mutated residues in Geminin^AWA, Geminin^RTGG and Geminin^KKFEV are indicated.

Figure 2

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.

Figure 3

Geminin^AWA inhibits MCM loading in vitro but not in vivo. (A) Geminin^AWA inhibits the MCM ←→Cdt1 interaction in vitro. Increasing concentrations of recombinant Geminin protein (5 μg/mL, 10 μg/mL, or 40 μg/mL) were mixed with translated Myc-Cdt1. After 40 min, purified MCM4/6/7 complex was added. After another 45 min, the reaction was incubated with anti-Myc antibody, and the precipitate was blotted for the proteins shown. (B, C) Geminin^AWA does not inhibit MCM loading in vivo. (B) Replication extracts were depleted of endogenous Geminin with anti-Geminin antiserum, then either recombinant Geminin^WT or Geminin^AWA was added back (50 μg/mL). Permeabilized sperm heads were added, and nuclei were allowed to assemble. The chromatin was then pelleted through a sucrose cushion, and the amount of MCM4 in the precipitate was determined by immunoblotting. (C) In a separate aliquot of the reaction, DNA replication was allowed to proceed to completion and the extent of replication was measured. Average of two experiments

Figure 4

Geminin^AWA does not inhibit re-replication. Replication extracts made from Xenopus eggs were depleted of both Geminin and Cdt1 using specific antibodies, then supplemented with recombinant Geminin:Cdt1 complexes. Replication was allowed to progress in the presence of BrdUTP, then the density of the product was determined by equilibrium ultracentrifugation. (A–D) Typical gradient profiles; (E) extent of re-replication for different Geminin mutants. The gray bar indicates the range, and the horizontal line indicates the mean. n, number of experiments.

Figure 5

Geminin^AWA does not inhibit HBO1 acetyltransferase activity. (A) Geminin^AWA forms a ternary complex with Cdt1 and HBO1. HA-Cdt1:FLAG-HBO1 complex was precipitated from HeLa cells expressing both proteins using anti-HA antibody. The precipitate was incubated with recombinant Geminin, and after extensive washing the amount of Geminin incorporated into the complex was determined by immunoblotting. (B) Geminin^AWA does not inhibit HBO1. HA-Cdt1:FLAG-HBO1 complex was purified using anti-HA antibody as in part (A), eluted with HA peptide, then incubated with acetyl coenzyme A, histone H4 peptide, and different recombinant Geminin proteins. The amount of acetylated histone H4 peptide was determined by immunoblotting.