The Inhibition Role of Geminin on Cdt1

As discussed above, during G1 phase, Cdc6 and Cdt1 are recruited by ORC to the replication origins and in turn required for the loading of MCM complex onto DNA to form the pre-RC. The formation of pre-RC is essential for the assembly of replication machinery and subsequent DNA replication initiation at S phase onset. Geminin starts to accumulate in the nucleus at this point of the cell cycle after DNA replication is initiated, and maintains its nuclear presence until the end of mitosis. Geminin physically interacts with Cdt1, and sequesters Cdt1 from its role in the pre-RC assembly, thus preventing DNA rereplication. In the cell-free Xenopus egg extracts system, the supply of recombinant Geminin protein disrupts the pre-RC assembly by inhibiting the loading of MCM proteins, without affecting the chromotin association of ORC and Cdc6. Consequently, this unloading of MCM proteins leads to an inhibition of nuclear DNA replication. All these inhibitory effects resulted from recombinant Geminin can be efficiently rescued by the addition of excess Cdt1 (McGarry and Kirschner, 1998; Wohlschlegel et al., 2000). Immunodepletion of endogenous Geminin from metaphase Xenopus egg extracts allows MCM complex to associate with replication origins and promotes DNA replication. This gain of licensing activity in the absence of Geminin is not due to the loss of CDK activity.

Conversely, the inhibition of CDKs in metaphase extracts stimulates origin assembly only after the depletion of Geminin, further suggesting that Geminin is the major inhibitor of rereplication in metaphase (Tada et al., 2001).

The inhibition of rereplication by Geminin through Cdt1 sequestration is conserved in metazoans. The Drosophila Geminin homolog also inhibits DNA replication in vitro by preventing binding of MCMs to chromatin (Quinn et al., 2001). The in vivo overexpression of Geminin in Drosophila embryos results in a general decrease of BrdU-labeling cells in mitotic and endoreplicating tissues, that is, an inhibition of DNA replication. The DNA replication inhibition phenotype is accompanied by a dramatic decrease of S phase cells, increased numbers of metaphase cells and apoptosis. In addition, Geminin overexpression during the early proliferative phase of the eye-antennal imaginal disc also results in an extraordinary decrease of S phase cells, thus the size of third instar larvae eye discs and adult eye. In agreement with the overexpression phenotypes and the inhibitory role of Geminin in DNA replication, Drosophila Geminin mutants exhibit overreplication defects late in embryogenesis and in oogenesis. For an instance, in most stage-12 wild type ovaries, DNA amplification is only observed in the anterior region in one focus per cell, whereas 100% of Geminin mutant stage-12 ovaries show strong BrdU labeling of four amplification loci in all follicle cells. By stage-14, all follicle cells of wild type ovaries have ceased DNA amplification, whereas many follicle cells from Geminin mutant ovaries still continue amplification. Mechanistically, Geminin not only physically associates with Cdt1 like in other species, which is clarified by an immunoprecipitation from Drosophila embryos, but also interacts genetically with Cdt1. A similar phenotype of Geminin overexpression is observed in Cdt1 mutants, suggesting their opposite roles in regulating DNA replication. Halving the dosage of Cdt1 enhances the Geminin overexpression eye phenotype, leading to a smaller and rougher eye. Vice versa, the Cdt1 mutant phenotypes can be suppressed by a Geminin mutant (Quinn et al., 2001). Therefore, it seems that the regulatory role of Geminin in DNA replication and the mechanism are conserved among metazoans.

Direct elimination of Geminin by antisense techniques in developing Xenopus embryos provide further insight into the roles and mechanisms of Geminin in preventing rereplication. The Geminin eliminated embryos have a unique early embryonic lethal phenotype. These embryos arrest in G2 phase immediately after the midblastula transition, the point in development when the cell cycle slows and zygotic gene expression begins. The cells in the deficient embryos show overreplicated DNA content, which confirms the role of Geminin in preventing rereplication. The mechanisms of Geminin loss of function to arrest the embryos in G2 phase partly

assign to a hyperphosphorylation of Chk1 protein kinase, an effector to implement checkpoint response. The activated Chk1 by increased phosphorylation prevents entry into mitosis in part by inhibiting Cdc25C, the phosphatase that removes the phosphates from T14 and Y15 of the mitotic cyclin-dependent kinase Cdc2 and consequently activates Cdc2 at the onset of mitosis. Cdc2 is always hyperphosphorylated on Y15 and maintained as an inactive form in the absence of Geminin. Bypassing of Chk1 pathway by injecting either Cdc25C mutant or Chk1 mutant individually rescues G2 arrest phenotype (McGarry, 2002). Similarly, in support to the rereplication inhibition role of Geminin, silencing of Geminin by siRNA in Drosophila Schneider D2 cells leads to a cessation of mitosis and asynchronous overrreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content (Mihaylov et al., 2002). This phenotype of Geminin deficiency is completely rescued by cosilencing of Cdt1, in agreement with the mechanism discussed above that Geminin prevents rereplication by direct sequestering Cdt1. In addition, the phenotype induced by Geminin knock down is also partially rescued by coablation of Chk1, again indicating the involvement of Chk1 in the checkpoint control in response to DNA overreplication (Mihaylov et al., 2002). Together, loss of function phenotypes in both Xenopus embryos and Drosophila cells reinforce our understandings that Geminin plays a pivotal role in DNA rereplication inhibition through Cdt1 binding and sequestration.

The Cdt1 interaction domain is mapped into the coiled-coil domain of Geminin. A fragment of XlGeminin consisting only of amino acids 87-168, which includes the coiled-coil domain, is sufficient to inhibit DNA replication (McGarry and Kirschner, 1998). By contrast, the N-terminal domain, which does not interact with Cdt1 but accounts for the neuralizing activity of Geminin (see below), has no effect on DNA replication or cell cycle progression. In mammals, the Geminin binding region of Cdt1 has also been characterized. The Cdt1 central region (amino acids 177-380) is demonstrated to be the Geminin binding domain, whereas Mcm6 interacts with the Cdt1 C-terminal region (amino acids 407-477). Interestingly, the C-terminal region of Cdt1 is conserved among all eukaryotes including yeast, whereas the central Geminin binding region is only conserved in metazoans, which exactly correlates with that Geminin is a metazoan specific protein.

Recent studies provide further insight into the molecular basis of the Geminin-Cdt1 regulatory mechanisms in mammalian cells. The DNA binding domain of Geminin-Cdt1

partly overlaps with its Geminin association domain. Therefore, the tight Geminin-Cdt1 interaction masks the DNA binding region of Geminin-Cdt1 and inhibits the association of Cdt1 to replication origins in the chromatin. Furthermore, the Geminin-Cdt1 interaction blocks the binding of Cdt1 to Mcm2/6 as well as Cdc6, thus inhibiting the association of Cdt1 into pre-RC and the subsequent MCM complex recruitment.

Although the Geminin and MCM interaction domains of Cdt1 are independent and the mechanisms underlying the inhibition of Cdt1-MCM association by Geminin still remain unclear, it is plausible to speculate that the binding of Geminin to the Cdt1 central region leads to a conformational change in the overall structure of Cdt1, which concomitantly results in its C-terminal MCM binding domain to be masked. Together, by means of interacting with and sequestering Cdt1, Geminin blocks the bindings of Cdt1 to DNA, Cdc6 and MCM proteins, thus inhibiting the assembly of pre-RC and preventing DNA rereplication (Yanagi et al., 2002; Cook et al., 2004). In addition, the CDK dependent phosphorylation of Cdt1 during S phase does not interfere with its binding to Geminin, suggesting that the phosphorylation and Geminin binding of Cdt1 are independent but function synergically to ensure a thorough inactivation of Cdt1, thus a complete DNA rereplication inhibition (Sugimoto et al., 2004).