Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.
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.
Gillespie
P J
PJ
CRC Chromosome Replication Research Group, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, UK. peter.gillespie@cshl.org
Li
A
A
Blow
J J
JJ
D000818:Animals; D018797:Cell Cycle Proteins / Q000494:pharmacology / Q000502:physiology; D002467:Cell Nucleus / Q000235:genetics*; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000502:physiology; D064248:Geminin; D008297:Male; D008957:Models, Genetic; D009711:Nucleotides / Q000494:pharmacology; D010063:Ovum / Q000737:chemistry; D018741:Replication Origin; D013094:Spermatozoa / Q000648:ultrastructure; D014981:Xenopus; D029867:Xenopus Proteins / Q000302:isolation & purification / Q000502:physiology*
C406819:Cdt1 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C577998:GMNN protein, Xenopus; D064248:Geminin; D009711:Nucleotides; D029867:Xenopus Proteins; D004247:DNA