Dynamic interactions of high Cdt1 and geminin levels regulate S phase in early Xenopus embryos

Cdt1 plays a key role in licensing DNA for replication. In the somatic cells of metazoans, both Cdt1 and its natural inhibitor geminin show reciprocal fluctuations in their protein levels owing to cell cycle-dependent proteolysis. Here, we show that the protein levels of Cdt1 and geminin are persistently high during the rapid cell cycles of the early Xenopus embryo. Immunoprecipitation of Cdt1 and geminin complexes, together with their cell cycle spatiotemporal dynamics, strongly supports the hypothesis that Cdt1 licensing activity is regulated by periodic interaction with geminin rather than its proteolysis. Overexpression of ectopic geminin slows down, but neither arrests early embryonic cell cycles nor affects endogenous geminin levels; apparent embryonic lethality is observed around 3-4 hours after mid-blastula transition. However, functional knockdown of geminin by ΔCdt1_193-447, which lacks licensing activity and degradation sequences, causes cell cycle arrest and DNA damage in affected cells. This contributes to subsequent developmental defects in treated embryos. Our results clearly show that rapidly proliferating early Xenopus embryonic cells are able to regulate replication licensing in the persistent presence of high levels of licensing proteins by relying on changing interactions between Cdt1 and geminin during the cell cycle, but not their degradation.     

Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.     

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.     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

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.     

Geminin has dimerization, Cdt1-binding, and destruction domains that are required for biological activity

Geminin is an unstable regulatory protein that affects both cell division and cell differentiation. Geminin inhibits a second round of DNA synthesis during S and G(2) phase by binding the essential replication protein Cdt1. Geminin is also required for entry into mitosis, either by preventing replication abnormalities or by down-regulating the checkpoint kinase Chk1. Geminin overexpression during embryonic development induces ectopic neural tissue, inhibits eye formation, and perturbs the segmental patterning of the embryo. In order to define the structural and functional domains of the geminin protein, we generated over 40 missense and deletion mutations and tested their phenotypes in biological and biochemical assays. We find that geminin self-associates through the coiled-coil domain to form dimers and that dimerization is required for activity. Geminin contains a typical bipartite nuclear localization signal that is also required for its destruction during mitosis. Nondegradable mutants of geminin interfere with DNA replication in succeeding cell cycles. Geminin's Cdt1-binding domain lies immediately adjacent to the dimerization domain and overlaps it. We constructed two nonbinding mutants in this domain and found that they neither inhibited replication nor permitted entry into mitosis, indicating that this domain is necessary for both activities. We identified several missense mutations in geminin's Cdt1 binding domain that were deficient in their ability to inhibit replication yet were still able to allow mitotic entry, suggesting that these are separate functions of geminin.     

Coenzyme Q10 as a potent compound that inhibits Cdt1-geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we found that coenzyme Q(10) (CoQ(10)) can inhibit Cdt1-geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ(10), having a longer isoprenoid chain, was the strongest inhibitor of Cdt1-geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 muM. Surface plasmon resonance analysis demonstrated that CoQ(10) bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ(10) had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ(10) inhibits Cdt1-geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ(10) was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ(10) could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1-geminin binding. The application of inhibitors for the formation of Cdt1-geminin complex is discussed.     

The inhibitory action of SQDG (sulfoquinovosyl diacylglycerol) from spinach on Cdt1-geminin interaction

A human replication initiation protein, Cdt1, is a central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by direct binding. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance, for example, by geminin silencing with small interfering RNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. We established a high throughput screening system based on a modified enzyme linked immunosorbent assay to identify compounds that interfere with human Cdt1-geminin binding. Using this system, we screened inhibitors from natural materials containing food components, and found that a glycolipid, sulfoquinovosyl diacylglycerol (SQDG), from spinach can inhibit Cdt1-geminin interaction in vitro, with 50% inhibition observed at concentrations of 1.79mug/ml. Other major glycolipids, such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) from spinach, had no influence. Surface plasmon resonance analysis demonstrated that SQDG bound selectively to Cdt1, but did not interact with geminin. Using three-dimensional computer modeling analysis, SQDG was considered to interact with the geminin interaction interface on Cdt1, and the sulfate group of SQDG was assumed to make hydrogen bonds with the residue of Arg346 of Cdt1. These data could help to further understanding of the structure and function of Cdt1. In addition, SQDG could be a clue to developing more appropriate inhibitors of Cdt1-geminin interactions.     

The replicative regulator protein geminin on chromatin in the HeLa cell cycle

Geminin is believed to have a major function in the regulation of genome replication and cell proliferation. Published evidence shows that geminin specifically interacts with Cdt1 to block its function in the assembly of prereplication complexes. However, in proliferating HeLa cells geminin and Cdt1 are co-expressed during a relatively short time at the G(1)-to-S phase transition. Under these conditions, nearly all Cdt1 and a major part of geminin are bound to chromatin and reside at the same or closely adjacent sites as shown here by chromatin immunoprecipitation. Cdt1 is rapidly degraded early in S phase, but geminin remains bound to the chromatin sites. One function that chromatin-bound geminin could perform is to prevent access to Cdt1 that may escape S phase-dependent degradation or is synthesized in excess. Indeed, Cdt1 continues to be synthesized in HeLa cells in S phase but never accumulates because of the efficient degradation. Therefore, geminin can be eliminated by RNA interference without detectable effects on cell cycle parameters.     

A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus

Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.     

Repression of Nascent Strand Elongation by Deregulated Cdt1 during DNA Replication in Xenopus Egg Extracts

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.     

Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G(1)-to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G(1), and with geminin in post-G(1) cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G(1) cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.     

Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) are removed, thus preventing the origin from firing again. Here we report the purification of the RLF-B component of the licensing system and show that it corresponds to Cdt1. RLF-B/Cdt1 was inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allowed them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulated origin assembly only after the depletion of geminin. These experiments suggest that geminin-mediated inhibition of RLF-B/Cdt1 is essential for repressing origin assembly late in the cell cycle of higher eukaryotes.     

Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity

DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.     

Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.     

The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells

Chromosomal DNA replication requires the recruitment of the six-subunit minichromosome maintenance (Mcm) complex to chromatin through the action of Cdc6 and Cdt1. Although considerable work has described the functions of Cdc6 and Cdt1 in yeast and biochemical systems, evidence that their mammalian counterparts are subject to distinct regulation suggests the need to further explore the molecular relationships involving Cdc6 and Cdt1. Here we demonstrate that Cdc6 and Cdt1 are mutually dependent on one another for loading Mcm complexes onto chromatin in mammalian cells. The association of Cdt1 with Mcm2 is regulated by cell growth. Mcm2 prepared from quiescent cells associates very weakly with Cdt1, whereas Mcm2 from serum-stimulated cells associates with Cdt1 much more efficiently. Cdc6, which normally accumulates as cells progress from quiescence into G(1), is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and we provide evidence that the mechanism of this inhibition involves blocking the binding of Cdt1 to both Mcm2 and Cdc6. These results identify novel molecular functions for both Cdc6 and geminin in controlling the association of Cdt1 with other components of the replication apparatus and indicate that the association of Cdt1 with the Mcm complex is controlled as cells exit and reenter the cell cycle.     

Geminin-Cdt1 balance is critical for genetic stability

A cell limits its DNA replication activity to once per cell division cycle to maintain its genomic integrity. Studies in a variety of organisms are elucidating how these controls are exercised. Key amongst these is the regulation of replication initiator proteins such as Cdt1. Cdt1 is present in cells in G1 phase where it is required for initiation of replication. Once origins have fired, Cdt1 is either exported out of the nucleus or degraded, thereby preventing another round of replication. Higher eukaryotes have evolved another redundant mechanism, an inhibitor called geminin, to restrain Cdt1 activity. Studies in multiple organisms have shown that unregulated Cdt1 activity stimulates overreplication of the genome. Interestingly, the same seems to be true when geminin is depleted. The imbalance in the activities of these proteins causes the activation of key checkpoint proteins, the ATM/ATR kinases and the tumor suppressor, p53. This review proposes that a balance between Cdt1 and geminin is important for maintaining genomic stability.