Activity of the purified proteins. Different quantities of purified proteins were subjected to specific individual assays, using Xenopus sperm nuclei as substrate. Protein quantity is given as mass of protein per ng template DNA (a-f) or mass of protein per μl extract (g). DNA synthesis is expressed as % of the DNA template replicated (a-f), or ng DNA synthesised per μl extract (g). a, Nucleoplasmin activity required in a crude licensing assay. b, ORC activity required for licensing in an ORC-depleted extract. c, Cdc6 activity required for licensing in an Cdc6-depleted extract. d, e, RLF-B activity required in a crude licensing assay. f, RLF-M/Mcm2-7 activity required in an Mcm2-7-depleted extract. g, Replication inhibition by geminin in whole egg extract containing 3 ng/μl DNA. Arrows show the protein quantity used in subsequent reconstitution experiments.