Stages in the assembly of licensed replication origins on sperm nuclei. A cartoon of a small segment of Xenopus sperm DNA is shown following exposure to egg cytoplasm. Nucleotide requirements are shown on the right. a, Condensed sperm chromatin, with H2A and H2B replaced by protamines. b, Removal of protamines by nucleoplasmin. c, Assembly of ORC onto origins, requiring ADP, ATP or ATP-γ-S. d, Assembly of Cdc6 and RLF-B/Cdt1 onto ORC-containing DNA, requiring ATP or ATP-γ-S. e, The loading of Mcm2-7 hetero-hexamers onto chromatin represents origin licensing. 10–20 copies of Mcm2-7 can be assembled onto each origin. This step requires hydrolysable ATP. f, Once licensing is complete, the affinity of Cdc6 and RLF-B/Cdt1 for origins is decreased (licensing-dependent origin inactivation).