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