Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import - PubMed (original) (raw)
Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import
Ben Hodgson et al. Curr Biol. 2002.
Abstract
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.
Figures
Figure 1
DNA-dependent generation of an inhibitor of replication licensing. a, Whole extract or membrane-free extract was pre-incubated with or without sperm nuclei (10 ng DNA/μl) plus or minus 2.5 μM wild-type or T24N mutant Ran, or 200 μg/ml wheat germ agglutinin. The ability of the PIEs to inhibit licensing in untreated interphase extracts was measured. Dashed lines, background licensing of sperm nuclei incubated in buffer alone. b, Sperm nuclei were incubated for 30 min in interphase extract supplemented with either buffer or PIE. Chromatin was isolated and immunoblotted for Orc1, Cdc6, Mcm3, Cdt1 and geminin. c, PIE, nucleoplasmic extract and cytoplasmic extract were subject to serial 3-fold dilutions, then assayed for their ability to inhibit licensing of interphase extract. Dashed line, background licensing of sperm nuclei incubated in buffer alone. d, Cytoplasmic extract, nucleoplasmic extract and insoluble nuclear pellet were immunoblotted for Orc1, Cdc6, Cdt1, Mcm7, geminin and CDKs (PSTAIR).
Figure 2
The licensing inhibitor is geminin. a, The ability of various pre-incubated extracts to inhibit replication licensing was assayed. sperm nuclei were pre-incubated in interphase extract minus or plus 2.5 mM roscovitine (PIE, PIE+rosc.). Alternatively, sperm nuclei were pre-incubated in interphase extract previously depleted of with antibodies against Orc1, Cdc6, Mcm3, Mcm7, Cdc7 or geminin, or mock-depleted with non-immune antibodies (NI–), or were depleted of Cdt1 using geminin-coupled beads. Alternatively, PIE was prepared in normal interphase extract, and then depleted with either anti-geminin antibodies (PIE, geminin–) or non-immune antibodies (PIE, NI–). Inset: Immunoblot of chromatin-bound Mcm7 following 30 min incubation of sperm nuclei in PIE (lane 1), PIE + recombinant Cdt1 (lane 2) or PIE prepared without DNA (lane 3). b, Wild type and geminin-DEL (which lacks a functional destruction box) were incubated at 1.3 ng/μl for 45 min in metaphase extract released into interphase with CaCl2 (lanes 2 and 5) or interphase extract (lanes 3 and 6). Input levels of recombinant geminin are shown in lanes 1 and 4. Samples were analysed by SDS-PAGE and immun0blotted with anti-geminin antibodies (top panel) or anti-6xHis antibodies (bottom panel). c, Metaphase extract (M) plus or minus sperm nuclei (10 ng DNA/μl) was released into interphase by addition of CaCl2 and at the indicated times (in min) aliquots were taken and immunoblotted for endogenous geminin and Cdt1.
Figure 3
Geminin’s affinity for Cdt-1 changes during the cell cycle**.** a-d, Gel filtration of different extracts immunoblotted for geminin and Cdt1: metaphase extract (a), interphase extract (10 min after metaphase release) (b), pre-incubated extract plus sperm (10 ng DNA/μl, 90 min) (c) and pre-incubated extract minus sperm (no DNA, 90 min) (d). Migration of molecular weight markers (in kDa) is shown above. e, Pre-incubated extracts were prepared plus or minus 10ng DNA/μl at the indicate times; 6xhis-Cdt1 was added and incubated for 30 min, then pulled out on Ni-NTA agarose. Bead-bound Cdt1 and geminin were analysed by SDS-PAGE and immunoblotting.
Figure 4
Model for geminin regulation during the Xenopus embryonic cell cycle. Cartoon showing a small segment of chromatin containing a replication origin as it passes through the cell cycle. Hexagons labelled ‘M’ represent hexameric complexes of Mcm2-7. a, In metaphase geminin is both abundant and active, so is able to inhibit origin licensing by formation of a tight complex with Cdt1/RLF-B. ORC and Cdc6 do not bind tightly to DNA, probably due to high CDK levels. b, Upon metaphase release some of the geminin is degraded, but the remaining geminin is altered in some way that prevents it binding and inhibiting Cdt1/RLF-B. The loss of CDK activity allows ORC and Cdc6 to associate tightly with DNA. With all components of the licensing system active, Mcm2-7 is loaded onto origins. c, Following nuclear assembly, geminin is imported into a functional nucleus and becomes reactivated. This allows it to bind and inhibit Cdt1/RLF-B, thus preventing the relicensing of replicated origins.
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