Anatoliy Li

Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus

In late mitosis and G1, Mcm2–7 are assembled onto replication origins to ‘license’ them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors—the origin recognition complex, Cdc6 and Cdt1—are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase‐promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re‐licensing and re‐replication. The extent of re‐replication is limited by checkpoint kinases that are activated as a consequence of re‐replication itself. These results allow us to build a comprehensive model of how re‐replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a “permissive” heterotrimer and an “inhibitory” heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.

Functional domains of the Xenopus replication licensing factor Cdt1

During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.

The origin of CDK regulation

Cip/Kip proteins that inhibit cyclin-dependent kinase 2 (Cdk2) restrain the initiation of DNA replication. Degradation of a Xenopus Kip1 orthologue, Xic1, is dependent on its recruitment to replication origins. This ensures that activation of Cdk2 and subsequent initiation of replication is co-ordinately regulated at, and localized to, replication origins.

Negative regulation of geminin by CDK-dependent ubiquitination controls replication licensing.

The replication licensing system ensures the precise duplication of chromosomal DNA in each cell cycle. In metazoans, a small protein called geminin plays a central role in negatively regulating licensing late in the cell cycle. Recent work using Xenopus egg extracts shows how geminin activity is downregulated on exit from metaphase in a process that requires mitotic cyclin-dependent kinases (CDKs). Geminin is polyubiquitinated by the Anaphase Promoting Complex, but instead of being proteolysed - the normal fate of polyubiquitinated proteins - much of the geminin is deubiquitinated, leaving it inactive. These results suggest a simple model for how precise chromosome duplication is ensured in the Xenopus model system.

Evidence for a mammalian late-G1 phase inhibitor of replication licensing distinct from geminin or Cdk activity.

Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood. We have employed a hybrid mammalian / Xenopus egg extract replication system to dissect activities that inhibit replication licensing at different stages of the cell cycle in Chinese Hamster Ovary (CHO) cells. We find that soluble extracts from mitotic cells inhibit licensing through a combination of geminin and Cdk activities, while extracts from S-phase cells inhibit licensing predominantly through geminin alone. Surprisingly however, geminin did not accumulate until after cells enter S phase. Unlike extracts from cells in early G1 phase, extracts from late G1 phase and early S phase cells contained an inhibitor of licensing that could not be accounted for by either geminin or Cdk. Moreover, inhibiting cyclin and geminin protein synthesis or inhibiting Cdk activity early in G1 phase did not prevent the appearance of inhibitory activity. These results suggest that a soluble inhibitor of replication licensing appears prior to entry into S phase that is distinct from either geminin or Cdk activity. Our hybrid system should permit the identification of this and other novel cell cycle regulatory activities.