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

DOI: 10.1186/1471-2091-2-15

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Abstract:

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 occuring 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-γ-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-γ-S.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.During S phase of the eukaryotic cell division cycle the entire genome must be faithfully duplicated. The many thousands of replication forks involved in this process must be co-ordinated to ensure that, despite the very large quantities of DNA involved, no section of DNA is left unreplicated and no section of DNA is replicated more than once. Cells achieve this by dividing the process of replication origin activation into two distinct phases [1-3]. During late mitosis and early G1, proteins are assembled onto replication origins which culminates in the origin becoming 'licensed' for a single round of DNA replication by loading complexes of Mcm2-7 proteins [4-8]. In yeast, a 'pre-replicative complex' (pre-RC) forms a footprint over replication origins during G1 [9] which may well correspond to

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