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. 2004 Mar;6(3):260-7.
doi: 10.1038/ncb1100. Epub 2004 Feb 8.

Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination

Anatoliy Li  1 , J Julian Blow
Affiliations

Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination

Anatoliy Li et al. Nat Cell Biol. 2004 Mar.

Abstract

In late mitosis and G1, a complex of the essential initiation proteins Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other times licensing is inhibited by cyclin-dependent kinases (CDKs) and geminin, thus ensuring that origins fire only once per cell cycle. Here we show that, paradoxically, CDKs are also required to inactivate geminin and activate the licensing system. On exit from metaphase in Xenopus laevis egg extracts, CDK-dependent activation of the anaphase-promoting complex (APC/C) results in the transient polyubiquitination of geminin. This ubiquitination triggers geminin inactivation without requiring ubiquitin-dependent proteolysis, and is essential for replication origins to become licensed. This reveals an unexpected role for CDKs and ubiquitination in activating chromosomal DNA replication.

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Figures

Figure 1
Figure 1
Roscovitine inhibits licensing at concentrations where MPF is inhibited. A, Schematic picture showing events occurring after calcium release of metaphase-arrested Xenopus egg extracts. In the left hand panel, calcium addition causes inactivation of mitotic CDK activity, and activates the licensing system. When S phase CDKs are activated the licensed DNA can replicate. In the right hand panel, 6-DMAP is added to the metaphase extract before calcium. The licensing system fails to become active, so no DNA replication takes place. B, Metaphase extracts were supplemented with Xenopus sperm and the indicated concentrations of roscovitine. The percentage of sperm heads assembled into interphase nuclei possessing an intact nuclear envelope was measured. C, Metaphase extracts were incubated for 15 min plus and minus calcium and roscovitine as indicated, before being used for a histone H1 kinase assay. Roscovitine concentrations were: 10, 20, 50, 100, 200, 500 and 1000 μM. D, Metaphase extracts were supplemented with 3 mM 6-DMAP or various concentrations of roscovitine prior to addition of 0.3 mM calcium (“metaphase + 6-DMAP + Ca” or “metaphase + rosco. + Ca”). Alternatively, 6-DMAP or roscovitine were added after calcium addition (“metaphase + Ca + 6-DMAP” or “metaphase + Ca + rosco.”). Xenopus sperm were then added to the extract, and after incubation for 20 min the chromatin was isolated and immunoblotted for Mcm7.
Figure 2
Figure 2
6-DMAP and roscovitine prevent release of geminin from Cdt1. Metaphase extracts were supplemented with or without 3 mM 6-DMAP or 500 μM roscovitine or were immunodepleted of Cdk1, and were then supplemented with 0.3 mM CaCl2 and incubated for 15 min. Alternatively, metaphase extract was supplemented with 0.3 mM calcium, followed by addition of recombinant gemininDEL and incubation for 15 min. Metaphase extract without addition is also shown. A, Extracts were separated by gel filtration and fractions immunoblotted for Cdt1 and geminin. The migration of molecular weight markers (in kDa) is indicated above. ‘r-geminin’ indicates the migration of gemininDEL. B, Immunoblot of total Cdt1 and geminin in: 1, metaphase extract alone; 2, metaphase extract + calcium; 3, metaphase extract + 6-DMAP + calcium; 4, metaphase extract + roscovitine + calcium. Endogenous Xenopus geminin migrates as a doublet, presumably reflecting the existence of two closely related geminin genes (geminin-H and geminin-L24). C, Gel filtration fractions 3-5 from extract supplemented with 500 μM roscovitine prior to CaCl2 addition were pooled together. They were then immunodepleted with antibodies against geminin (“gem.-”), Cdt1 or non-immune antibodies (“NI-”). Immunodepleted extract was then separated by SDS PAGE and immunoblotted for geminin and Cdt1. The starting pool without any immunodepletion is also shown (“pool”).
Figure 3
Figure 3
The only inhibitory activity in treated extracts is geminin. A, Metaphase extract was supplemented with 3 mM 6-DMAP and then 0.3 mM calcium (“6-DMAP extract”). 6-DMAP extracts were immunodepleted with antibodies to geminin, or with non-immune antibodies. The resultant extracts were assayed for their ability to inhibit the licensing of interphase Xenopus extracts. Licensing is expressed as the % of that seen with no inhibitory extract. Dashed lines, background licensing of sperm nuclei incubated in buffer alone. B, Metaphase extract was supplemented with or without 3 mM 6-DMAP and then 0.3 mM calcium. Various concentrations of recombinant Cdt1 (1.8, 5.4 and 16 nM) were then added, and the extracts were assayed for their ability to replicate sperm nuclei. C, Metaphase extract was supplemented plus or minus 3 mM 6-DMAP or 500 μM roscovitine or 300 nM recombinant Cdt1, prior to addition of 0.3 mM calcium. Untreated metaphase extract is also shown. Sperm nuclei were then added to the extract, and after 20 min incubation chromatin was isolated and immunoblotted for Mcm7.
Figure 4
Figure 4
Inhibition of the APC/C prevents activation of the licensing system. Metaphase extracts were supplemented with D-box peptide (2 mM; A, C) or ubiquitin K48R (1.2 mM; B, C) then plus or minus 0.3 mM calcium as indicated. A, After incubation for 20 min, extracts were immunoblotted for cyclin B and geminin (bottom panels). Alternatively, sperm nuclei were incubated in the extracts for 20 min, chromatin was then isolated and immunoblotted for Mcm7 (top panel). B, After incubation for 20 min, extracts were immunoblotted for geminin. C, After incubation for 20 min extracts were gel filtered and immunoblotted for Cdt1 and geminin.
Figure 5
Figure 5
Proteasome-mediated proteolysis is not required for activation of licensing. A, Metaphase extracts were supplemented with various concentrations of MG-132 (0, 0.51, 0.256, 1, 6, 32, 160, 800 μmol) and then plus or minus 0.3 mM calcium. After incubation for 20 min, extracts were immunoblotted for cyclin B and geminin. B, Metaphase extracts were supplemented plus or minus 800 μM MG-132 or 0.3 mM calcium. Sperm nuclei were then added, and after incubation for 20 min, chromatin was isolated and immunoblotted for Mcm7. C, Metaphase extracts were supplemented with or without 800 μM MG-132 or 0.3 mM calcium. Extracts were then assayed for their ability to replicate sperm nuclei. D, Metaphase extracts were supplemented with epoxymicin (500 μmol) and then plus or minus 0.3 mM calcium. After incubation for 20 min, extracts were immunoblotted for cyclin B and geminin. E, Metaphase extracts were supplemented with MG-132 (800 μM) or epoxymicin (500 μM) and then 0.3 mM calcium, after which they were gel filtered and immunoblotted for Cdt1 and geminin.
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References

    1. Diffley JFX. Building the perfect switch. Current Biol. 2001;11:R367–370. - PubMed
    1. Blow JJ, Hodgson B. Replication licensing - defining the proliferative state? Trends Cell Biol. 2002;12:72–78. - PMC - PubMed
    1. Nishitani H, Lygerou Z. Control of DNA replication licensing in a cell cycle. Genes Cells. 2002;7:523–534. - PubMed
    1. Chong JP, Mahbubani HM, Khoo CY, Blow JJ. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995;375:418–421. - PubMed
    1. Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995;81:601–609. - PubMed

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