Gillespie PJ et al. (2001), Reconstitution of licensed replication origins ...

XB-ART-7993

BMC Biochem 2001 Jan 01;2:15. doi: 10.1186/1471-2091-2-15.

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Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.

Gillespie PJ , Li A , Blow JJ .

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BACKGROUND: In order to ensure precise chromosome duplication, eukaryotes "license" their replication origins during late mitosis and early G1 by assembling complexes of Mcm2-7 onto them. Mcm2-7 are essential for DNA replication, but are displaced from origins as they initiate, thus ensuring that no origin fires more than once in a single cell cycle. RESULTS: Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occurring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-gamma-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-gamma-S. CONCLUSIONS: Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.

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Species referenced: Xenopus
Genes referenced: cdc6 cdt1 gmnn h2ac21 h2bc21 mcm2 mcm3 mcm7 mmut npm1 orc1 orc6 rlf

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	Figure 1. Fractionation scheme for proteins used in this study. Interphase Xenopus egg extract was fractionated by the methods outlined. See Materials and Methods for more details. Cdc6 was not purified to homogeneity but was replaced in the reconstituted reaction with recombinant material produced in insect cells.
	Figure 2. Purified proteins used in this study. Purified proteins were run on SDS polyacrylamide gels, and visualized with either Coomassie Blue (a-c, e-h) or silver staining (d). a, Nucleoplasmin purified from Xenopus eggs. b, ORC purified from Xenopus eggs. c, Recombinant Xenopus Cdc6 purified from baculovirus-infected insect cells. d, RLF-B/Cdt1 purified from Xenopus eggs. e, Recombinant Xenopus ΔCdt1 purified from E. coli.f, Mcm(2+4+6+7) purified from Xenopus eggs. g, Mcm(3+5) purified from Xenopus eggs. h, Recombinant Xenopus geminin purified from E. coll.
	Figure 3. 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.
	Figure 4. Reconstitution of licensing using purified proteins. a, c, d, Licensing activity of different combinations of purified proteins incubated in the presence of 2.5 mM ATP. DNA synthesis is expressed as % of the DNA template replicated. b, Western blot of Mcm7 loaded onto sperm chromatin in whole Xenopus extract, the full reconstituted reaction and the reconstituted reaction lacking RLF-B/Cdt1. The reconstituted reactions contained 2.5 mM ATP.
	Figure 5. Requirements for Cdt1 and geminin association with chromatin. a, b,Xenopus sperm nuclei were incubated with the indicated proteins in the presence of 2.5 mM ATP. Chromatin was then isolated and immunoblotted for the presence of bound Orc1, Cdt1 and geminin. The RLF-B/Cdt1 was a slightly more active fraction taken from the geminin bead eluate.
	Figure 6. 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.
	Figure 7. Nucleotide requirement for Cdc6 chromatin association in desalted extracts. Sperm nuclei were incubated in desalted whole egg extract 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 Cdc6.
	Figure 8. 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).

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