Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report t he purification of the RLF-B component of the licensing system and show tha t it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein th at is degraded during late mitosis. Immunodepletion of geminin from metapha se extracts allowed them to assemble licensed replication origins. Inhibiti on of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cyc le of higher eukaryotes.