Figure 6
From: Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins
Nucleotide requirements of the reconstituted reaction. Xenopus sperm nuclei were incubated with nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1, Mcm2-7 plus or minus geminin in the presence of either ADP, ATP, ATP-γ-S at 2.5 mM, or no added nucleotide. a, The degree of functional licensing was assessed, expressed as % of the DNA template replicated. b, Chromatin was isolated and immunoblotted for the presence of Orc1, Cdt1, Mcm3 and geminin. The Mcm2-7 was a slightly more active fraction taken from the gel filtration step in the purification, and the RLF-B/Cdt1 was from the geminin bead eluate.