Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.
During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.
Hodgson
Ben
B
Cancer Research UK Chromosome Replication, Research Group, Wellcome Trust Biocentre, University of Dundee, Dow Street, DD1 5EH, Dundee, United Kingdom.
Li
Anatoliy
A
Tada
Shusuke
S
Blow
J Julian
JJ
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D064248:Geminin; D008297:Male; D008954:Models, Biological; D010063:Ovum / Q000378:metabolism; D011485:Protein Binding; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D014981:Xenopus / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; C577998:GMNN protein, Xenopus; D064248:Geminin; D029867:Xenopus Proteins