double parked


Double parked homologs in yeast

An immunoprecipitation-PCR cycle has been used to isolate physically genomic DNA sequences that are bound by the fission yeast cdc10 gene product (involved in transcriptional activation at the start of the DNA synthetic phase) in an attempt to identify novel target genes. An essential gene, cdt1, has been isolated whose expression is cell cycle regulated in a cdc10 dependent manner. The cdt1 promoter contains a recognition site for a sequence specific DNA binding factor. The cdc10 gene product is a component of this factor. Ectopic expression of cdt1 can complement a temperature sensitive mutation of cdc10 at semipermissive temperature. Cells carrying a null allele of cdt1 are defective in DNA replication but initiate mitotic events, suggesting that cdt1 is essential for the normal dependency relationship of S-phase and mitosis (Hofmann, 1994).

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast: Drosophila homolog -- Cdc6)) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18, which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and a re-replication assay has been used to identify Cdt1 as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants, suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes (Nishitani, 2000).

Faithful duplication of the genetic material requires that replication origins fire only once per cell cycle. Central to this control is the tightly regulated formation of prereplicative complexes (preRCs) at future origins of DNA replication. In all eukaryotes studied, this entails loading by Cdc6 of the Mcm2-7 helicase next to the origin recognition complex (ORC). More recently, another factor, named Cdt1, was shown to be essential for Mcm loading in fission yeast and Xenopus as well as for DNA replication in Drosophila and humans. Surprisingly, no Cdt1 homolog was found in budding yeast, despite the conserved nature of origin licensing. This study identifies Tah11/Sid2, previously isolated through interactions with topoisomerase and Cdk inhibitor mutants, as an ortholog of Cdt1. sid2 mutants lose minichromosomes in an ARS number-dependent manner, consistent with ScCdt1/Sid2 being involved in origin licensing. Accordingly, cells partially depleted of Cdt1 replicate DNA from fewer origins, whereas fully depleted cells fail to load Mcm2 on chromatin and fail to initiate but not elongate DNA synthesis. It is concluded that origin licensing depends in S. cerevisiae as in other eukaryotes on both Cdc6 and Cdt1.

Cdt1 is essential for loading Mcm2-7 proteins into prereplicative complexes (pre-RCs) during replication licensing and has been found in organisms as diverse as fission yeast and humans. A homologue of Cdt1 has been identified in Saccharomyces cerevisiae, that is required for pre-RC assembly. Like Mcm2-7p, Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by cyclin dependent kinases (cdks). Cdt1p interacts with the Mcm2--7p complex, and the nuclear accumulation of these proteins during G1 is interdependent. This coregulation of Cdt1p and Mcm2-7p represents a novel level of pre-RC control (Tanaka, 2002).

In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. Helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an 'RXL' cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition (Chen, 2011).

Double parked/CDT-1 homologs in C. elegans

Genome stability requires that genomic DNA is replicated only once per cell cycle. The replication-licensing system ensures that the formation of prereplicative complexes is temporally separated from the initiation of DNA replication. The replication-licensing factors Cdc6 and Cdt1 are required for the assembly of prereplicative complexes during G1 phase. During S phase, metazoan Cdt1 is targeted for degradation by the CUL4 ubiquitin ligase (see Drosophila Cul4), and vertebrate Cdc6 is translocated from the nucleus to the cytoplasm. However, because residual vertebrate Cdc6 remains in the nucleus throughout S phase, it has been unclear whether Cdc6 translocation to the cytoplasm prevents rereplication. The inactivation of C. elegans CUL-4 is associated with dramatic levels of DNA rereplication. This study shows that C. elegans CDC-6 is exported from the nucleus during S phase in response to the phosphorylation of multiple CDK sites. CUL-4 promotes the phosphorylation and subsequent translocation of CDC-6 via negative regulation of the CDK-inhibitor CKI-1. Rereplication can be induced by coexpression of nonexportable CDC-6 with nondegradable CDT-1, indicating that redundant regulation of CDC-6 and CDT-1 prevents rereplication. This demonstrates that CDC-6 translocation is critical for preventing rereplication and that CUL-4 independently controls both replication-licensing factors (Kim, 2007).

In humans and Xenopus, ectopically expressed Cdc6 is completely exported from the nucleus during S phase; in contrast, a substantial fraction of endogenous Cdc6 remains nuclear localized during S phase. Strikingly, a similar result is observed in C. elegans, with a substantial fraction of endogenous CDC-6 remaining in the nucleus during S phase, whereas ectopically expressed CDC-6 appears exclusively cytoplasmic. The reason(s) for these differential localizations are not understood (Kim, 2007).

The presence of nuclear-localized Cdc6 during S phase in mammalian cells has led to the proposal that Cdc6 translocation is not important for restraining DNA-replication licensing. Further, there is currently no evidence for a functional role of Cdc6 translocation in preventing rereplication. In this study, it was observed that nonexportable CDC-6 can synergize with deregulated CDT-1 to induce rereplication. This implies that CDC-6 translocation is a redundant safeguard to prevent the reinitiation of DNA replication. This provides the first evidence in any organism of a functional role for phosphorylation-dependent CDC-6 nuclear export (Kim, 2007).

In S. pombe, the overexpression of the Cdc6 ortholog (Cdc18) is sufficient to induce significant rereplication. In contrast, overexpression of Cdc6 does not induce rereplication in S. cerevisiae, Drosophila, or humans. In humans, co-overexpression of wild-type Cdt1 and Cdc6 in cells that lack a cell-cycle checkpoint produces only modest rereplication in a subset of cells (Kim, 2007).

Coexpression of nondegradable CDT-1 and nonexportable CDC-6 produces significant rereplication in a subset of early-stage C. elegans embryos. In contrast, overexpression of combinations of deregulated and wild-type CDT-1 or CDC-6 does not induce rereplication. This indicates that redundant regulation of CDT-1 and CDC-6 prevents rereplication. No rereplication was observed in every embryonic cell expressing deregulated CDT-1 and CDC-6. This suggests that there might be additional mechanisms that act in the early embryo to limit rereplication (Kim, 2007).

The expression of combinations of wild-type and deregulated CDT-1 and CDC-6 produced an embryonic lethality that was not associated with increased DNA levels. The cause of this lethality is unclear, but it might arise from changes in DNA-replication timing, which is known to produce embryonic arrest (Kim, 2007).

Inactivation of CUL-4 produces dramatic levels of rereplication that are associated with a failure to degrade CDT-1. However, overexpressing Cdt1 in fission yeast does not induce rereplication, and overexpressing human Cdt1 several-log-fold higher than the endogenous protein produces only modest rereplication in a subset of cells. Given the negligible or limited effects of greatly overexpressing Cdt1 in other organisms, it was hard to reconcile the substantial rereplication associated with merely failing to degrade CDT-1 during S phase in cul-4(RNAi) animals (Kim, 2007).

This work reveals that the CDC-6 replication-licensing factor is also deregulated in cul-4(RNAi) animals. CDC-6 remains nuclear throughout S phase in cul-4(RNAi) animals, and this is correlated with a failure to phosphorylate CDC-6 on CDK sites. CUL-4 negatively regulates the levels of the CDK inhibitor CKI-1. The negative regulation of CKIs of the CIP/KIP family by CUL-4 is conserved in Drosophila and humans. cki-1 RNAi suppresses rereplication in cul-4 mutants without affecting CDT-1 accumulation, indicating that CKI-1 is independently required for the induction of rereplication. Significantly, the presence of CKI-1 is required for the block on CDC-6 phosphorylation and nuclear export in cul-4(gk434) cells. These results suggest that CUL-4 promotes CDC-6 nuclear export by negatively regulating CKI-1 levels, thereby allowing CDK(s) to phosphorylate CDC-6 and induce its nuclear export. The evidence that CDK(s) are the relevant kinases is that CDC-6 is phosphorylated on CDK consensus sites and the phosphorylation is blocked by a CDK inhibitor. In yeast and mammals, CDK activity prevents rereplication, and siRNA codepletion of CDK1 and CDK2 in human cells induces limited rereplication. These results suggest that in metazoa, Cdc6 is one of the critical targets of CDKs for preventing rereplication. This work further indicates that CUL-4 is a master regulator that restrains DNA replication through two independent pathways: mediating CDT-1 degradation and promoting CDC-6 nuclear export via the negative regulation of CKI-1 (Kim, 2007).

Double parked homologs in Xenopus

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6, a protein that allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. A protein that is required for the assembly of pre-replication complexes has been identified in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1, a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Using Xenopus in vitro systems, it has been shown that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase (Maiorano, 2000).

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. Geminin does not interfere with the chromatin association of Cdt1 and 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. Loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. Geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes; binding overlaps 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, 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 (Maiorano, 2004).

In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. This study shows that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. The results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase (Arias, 2004).

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. This study examines how licensing is regulated in Xenopus egg extracts. Cdt1 is shown to be 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 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 the building of 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 (Li, 2005).

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. This study shows 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. The 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 (Kisielewska, 2011).

Association of Geminin with Cdt1

In all eukaryotic organisms, inappropriate firing of replication origins during the G2 phase of the cell cycle is suppressed by cyclin-dependent kinases. Multicellular eukaryotes contain a second putative inhibitor of re-replication, called geminin (see Drosophila Geminin). Geminin is believed to block binding of the mini-chromosome maintenance (MCM) complex to origins of replication, but the mechanism of this inhibition is unclear. Geminin is shown to interact tightly with Cdt1, a recently identified replication initiation factor necessary for MCM loading. The inhibition of DNA replication by geminin that is observed in cell-free DNA replication extracts is reversed by the addition of excess Cdt1. In the normal cell cycle, Cdt1 is present only in G1 and S, whereas geminin is present in S and G2 phases of the cell cycle. Together, these results suggest that geminin inhibits inappropriate origin firing by targeting Cdt1 (Wohlschlegel, 2000).

Eukaryotic replication origins are 'licensed' for replication early in the cell cycle by loading Mcm(2-7) proteins. As chromatin replicates, Mcm(2-7) proteins are removed, thus preventing the origin from firing again. The purification of the RLF-B component of the licensing system has been purified; it corresponds to Cdt1. RLF-B/Cdt1 is inhibited by geminin, a protein that is degraded during late mitosis. Immunodepletion of geminin from metaphase extracts allows them to assemble licensed replication origins. Inhibition of CDKs in metaphase stimulates 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 (Tada, 2001).

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in G1, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. The human Cdt1 homolog (hCdt1), a nuclear protein, is present only during G1. After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G2. hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells (Nishitani, 2001).

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. How rereplication is prevented in normal mammalian cells has been investigated; these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 (Drosophila homolog: Double parked) and Cdc6 (Drosophila homolog: Origin recognition complex subunit 1) along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability (Vaziri, 2003).

To test whether geminin inhibits rereplication induced by Cdt1, geminin was overexpressed along with Cdt1 and Cdc6. Overexpression of geminin partially inhibits the rereplication mediated by Cdt1+Cdc6. Overexpression of Cdt1 (by itself) leads to a paradoxical increase in geminin levels in the rereplicating cells. In order to confirm that there was free Cdt1 (uncomplexed with geminin) in the cell lines, all the geminin was precleared from these cell extracts before immunoblotting for residual Cdt1 in the supernatant. The results show that despite the induction of geminin, not enough of the protein is produced to associate with and inhibit all the overexpressed Cdt1. The increase in geminin was attributed to a 10-fold induction of geminin mRNA seen upon overexpression of Cdt1. The mechanism of this induction is currently unclear but suggests the existence of a feedback loop between Cdt1 and its antagonist geminin (Vaziri, 2003).

The activation of the DNA damage checkpoint pathway and the tumor suppressor protein p53 provides a pathway by which mammalian cells prevent rereplication. Rereplication appears to lead to DNA damage. The data suggest that activation of ATM/ATR kinases caused by overexpression of Cdt1 and Cdc6 leads to direct phosphorylation of p53 and indirect phosphorylation of p53 through Chk2 kinase. Phosphorylation of p53 stabilizes the protein and leads to increased transcription and expression of p21. The latter is a potent inhibitor of cyclin A-cdk2 kinase and could therefore prevent any rereplication. Consistent with this hypothesis, overexpression of wild-type p53 or of p21 effectively inhibits rereplication in the p53-negative H1299 cells, while inactivation of p53 in A549 cells by overexpressing Mdm2 prevents p21 induction and permits rereplication. Because of the concurrent induction of proapoptotic genes like PIG3, p53 could also promote apoptosis of cells that have already undergone significant rereplication. Since mutations in p53 have been widely documented to promote genomic instability and gene amplification, these results provide a partial explanation of this observation by proposing a mechanism by which p53 stabilizes the genome. Genes other than p53, however, also prevent gene amplification, so it is unlikely that p53 is the only barrier to rereplication upon overexpression of Cdt1 and Cdc6 in all cell lines (Vaziri, 2003).

Geminin is an unstable inhibitor of DNA replication that negatively regulates the licensing factor CDT1 and inhibits pre-replicative complex (pre-RC) formation in Xenopus egg extracts. A novel function of Geminin is described. Human Geminin protects CDT1 from proteasome-mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle. Moreover, inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. These results demonstrate that Geminin is both a negative and positive regulator of pre-RC formation in human cells, playing a positive role in allowing CDT1 accumulation in G2-M, and preventing relicensing of origins in S-G2 (Ballabeni, 2004).

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, over 40 missense and deletion mutations were generated and their phenotypes were tested in biological and biochemical assays. Teminin self-associates through the coiled-coil domain to form dimers and 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. Two nonbinding mutants in this domain were constructed and they were found to neither inhibit replication nor permit entry into mitosis, indicating that this domain is necessary for both activities. Several missense mutations in geminin's Cdt1 binding domain were identified 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 (Benjamin, 2004).

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. The crystal structure of the mouse geminin-Cdt1 complex is described using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance (Lee, 2004).

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets (Saxena, 2004).

The APC/C inhibitor, Emi1, is essential for prevention of rereplication

Emi1 (early mitotic inhibitor) inhibits APC/C (anaphase-promoting complex/cyclosome) activity during S and G2 phases, and is believed to be required for proper mitotic entry. Emi1 plays an essential function in cell proliferation by preventing rereplication. Rereplication seen after Emi1 depletion is due to premature activation of APC/C that results in destabilization of geminin and cyclin A, two proteins shown in this study to play redundant roles in preventing rereplication in mammalian cells. Geminin is known to inhibit the replication initiation factor Cdt1. The rereplication block by cyclin A is mediated through its association with S and G2/M cyclin-dependent kinases (Cdks), Cdk2 and Cdk1, suggesting that phosphorylation of proteins by cyclin A-Cdk is responsible for the block. Rereplication upon Emi1 depletion activates the DNA damage checkpoint pathways. These data suggest that Emi1 plays a critical role in preserving genome integrity by blocking rereplication, revealing a previously unrecognized function of this inhibitor of APC/C (Machida, 2007).

DNA replication initiates from the specific regions of chromosomal DNA called origins. A critical question in the field of genomic stability is how cells manage to restrict the firing of origins to once and only once per cell cycle. Not only is this regulation achieved despite the fact that thousands of origins fire asynchronously all over the genome, but the regulation is selectively breached during certain stages of development as in endoreduplicating trophoblasts. Most of this regulation appears to be executed at the level of prereplicative complex (pre-RC) formation or origin licensing. The origin recognition complex (ORC) recruits Cdc6 and Cdt1 to origins in G1, which in turn load the putative replicative helicase, Mcm2-7, to form pre-RCs. After replication initiation in the subsequent S phase, Mcm2-7 are depleted from origins, but reformation of pre-RCs is prevented until chromosomes are segregated in M phase. Higher eukaryotes, including human cells, express geminin a protein that binds to Cdt1 to prevent loading of Mcm2-7 on post-initiation origins. Knockdown of geminin by RNA interference (RNAi) is sufficient to induce rereplication in many human cell lines, and geminin-null mice show enhanced endoreduplication in trophoblasts of early embryos. These results suggest that geminin is a major inhibitor of rereplication in mammalian cells. Yeast, however, do not encode geminin, and rereplication is prevented solely by the high levels cyclin-dependent kinase (Cdk) activity seen during S and G2 phases of the cell cycle. A role of high Cdk activity in rereplication block has also been suggested in human cells, although cancer cells that show rereplication after geminin knockdown do so without any obvious mechanism to concurrently inhibit Cdk2 activity. It is therefore currently unclear whether geminin and Cdk play redundant roles in rereplication block, or whether these mechanisms function in different parts of the cell cycle or in different tissues (Machida, 2007 and references therein).

The regulated degradation of proteins by proteasomes through a carefully orchestrated polyubiquitination program is a critical component of cell cycle regulation. For example, Cdks are important for progression through S, G2, and M, but their activity is regulated by the periodic accumulation and destruction of different types of cyclins. Levels of cyclins in the cell cycle are regulated by two ubiquitin ligases, SCF and anaphase-promoting complex/cyclosome (APC/C). SCF complex uses an F-box protein as a substrate recognition subunit. For example, SCFFBXW7 polyubiquitinates the G1 cyclin, cyclin E, for degradation in S phase. There are >60 F-box proteins in the human genome but the cellular functions of most of them are not yet known. APC/C, on the other hand, uses substrate recognition subunits Cdc20 and Cdh1 to polyubiquitinate substrates like cyclins A and B (and geminin) in mitosis and the subsequent G1. Cdc20 activates APC/C in mitosis while Cdh1 activates APC/C in late M and G1 phases. Both these subunits target substrates by recognizing a destruction motif called D-box, while Cdh1, in addition, recognizes a KEN-box (Machida, 2007 and references therein).

Since cyclin A/Cdk kinase activity is essential for DNA replication, the inactivation of APC/C at the G1/S transition is critical for the accumulation of cyclin A for S-phase progression. Emi1 (early mitotic inhibitor) is a cellular inhibitor of APC/C that is not present in yeasts, and is induced by the E2F transcription factor to inactivate APC/C at the G1/S transition. Emi1-mediated reduction in APC/C activity allows cells to accumulate cyclin A, and then the accumulated cyclin A-Cdk complex in S phase further suppresses APC/C activity by phosphorylating Cdh1. Because of its role in suppression of APC/C and because the latter is critical for mitosis, the primary role of Emi1 is believed to ensure proper mitotic entry. At the onset of mitosis, Emi1 is degraded following phosphorylation by Plk1 and ubiquitination by SCF-TrCP, and this allows the activation of APC/C that is critical for progression through M phase and the subsequent G1 phase (Machida, 2007 and references therein).

Since SCF is involved in many aspects of cell cycle regulation, a search was initiated for F-box proteins that are required for cell proliferation by RNAi screening. This screen identified Emi1, an F-box protein known as FBXO5, to be essential for cell proliferation. Surprisingly, the block to cell proliferation was accompanied by extensive rereplication after Emi1 depletion. This rereplication is due to premature activation of APC/C in S and G2 cells. Among many APC/C substrates, cyclin A and geminin are critical for prevention of rereplication, and the two proteins act simultaneously in S and G2 cells as redundant barriers to rereplication. These data suggest that an essential function of Emi1 in S and G2 cells is to prevent rereplication via stabilization of inhibitors of rereplication such as cyclin A and geminin (Machida, 2007).

Association of Cdt1 with minichromosome maintenance proteins and Cdc6

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. Cdc6 and Cdt1 are shown to be 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. Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region that is essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and evidence is provided 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 (Cook, 2004).

The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors, and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. This study investigated the function of the Saccharomyces cerevisiae Orc6, the only ORC subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits prereplicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. Two regions of Orc6 bind Cdt1 directly, and the extreme C terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of Mcm2-7 loading (Chen, 2007).

Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. Mcm2-7 loading was reconstituted with purified budding yeast proteins. Using biochemical approaches and electron microscopy, it was shown that single heptamers of Cdt1/Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. This work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication (Remus, 2009).

These results provide the first evidence that ORC and Cdc6 load the Mcm2-7 proteins from single Cdt1/Mcm2-7 heptamers into pre-RCs as head-to-head double hexamers. DNA, probably double stranded, passes through the long, central channel of this double hexamer. And, once loaded, the double hexamer is mobile, capable of passive one-dimensional diffusion or 'sliding' along DNA. These features of the pre-RC have implications for how origins are chosen and how replisomes assemble during initiation (Remus, 2009).

The loading of Mcm2-7 requires ORC, Cdc6, and hydrolysable ATP, consistent with requirements in vivo. The requirement for Cdt1 was not tested because it is an integral component of the Mcm2-7 complex. The interaction of Cdt1 with both Mcm2-7 and Orc6 suggests that it may act as a bridge between ORC and Mcm2-7. However, the results demonstrate that Cdc6 is also essential to recruit Mcm2-7 to origins, indicating that additional interactions are involved in this recruitment (Remus, 2009).

Surprisingly, loading of Mcm2-7 in vitro does not require specific ORC binding sites. The results may contribute to resolving the long-standing issue of how orthologs of ORC can act on specific DNA sequences in yeast, but show little or no sequence preference in metazoans. The results indicate that even yeast ORC has no inherent mechanistic requirement for specific DNA sequences in the loading of Mcm2-7. The sequence specific DNA binding of the budding yeast ORC may be an evolutionary adaptation designed to ensure sufficient origin activity in a genome containing very little intergenic DNA. Sequence specificity appears to be an integral part of the S. cerevisiae core ORC while sequence specificity of Schizosaccharomyces pombe ORC is conferred by an extended AT hook domain on the Orc4 subunit. Recruitment of ORC in metazoans may also involve interactions with additional sequence specific DNA binding proteins like TRF2. Consistent with this idea, recruitment of ORC to a GAL4 DNA binding site array via fusion of ORC subunits or Cdc6 to the GAL4 DNA binding domain is sufficient to create a functional replication origin in human cells (Remus, 2009 and references therein).

The binding of Mcm2-7 around double-stranded DNA has implications for how DNA unwinding is ultimately catalyzed by the Cdc45/Mcm2-7/GINS (CMG) complex. Mcm2-7 may act in unwinding analogously to the eukaryotic viral SF3 initiator/helicases including the SV40 large T antigen (TAg) and the papillomavirus E1 protein. The TAg double hexamer can bind to double-stranded DNA, and this binding can induce the generation of a short (8 bp) stretch of melted DNA specifically within one of the two hexamers. Although TAg and E1 can assemble as double hexamers around double-stranded DNA, current models indicate that they act during unwinding as classical helicases by encircling single-stranded DNA. If Mcm2-7 act analogously to these proteins, then CDK-and DDK-dependent events must promote remodeling of the Mcm2-7 complex to encircle single-stranded DNA during origin melting (Remus, 2009 and references therein).

Alternatively, Mcm2-7 may act during replication as a double-strand DNA translocase. In this model, Cdc45 and/or GINS would play a direct, structural role in strand separation, perhaps acting as a 'plough' or 'pin' into which DNA is pumped by Mcm2-7. This is analogous to the bacterial RuvAB Holliday junction branch migrating enzyme in which two RuvB hexamers pump double-stranded DNA through a tetramer of RuvA, which coordinates the separation and reannealing of strands. This second model is favored because it does not require topological reorganization of Mcm2-7 subunits during initiation and because it provides a potential biochemical function for Cdc45 and/or GINS during replication. The helicase activity of archaeal MCM as well as eukaryotic Mcm2-7 complexes on single-stranded DNA substrates need not reflect their mode of action in vivo: even double-stranded DNA translocases like RuvB can function in standard helicase assays, presumably because they can translocate along one strand of DNA and displace annealed oligonucleotides (Remus, 2009 and references therein).

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. This study established that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, it was shown that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, it was demonstrated that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. This study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation (Samel, 2014).

HBO1 histone acetylase is a coactivator of the replication licensing factor Cdt1

HBO1 histone acetylase is important for DNA replication licensing. In human cells, HBO1 associates with replication origins specifically during the G1 phase of the cell cycle in a manner that depends on the replication licensing factor Cdt1, but is independent of the Cdt1 repressor Geminin. HBO1 directly interacts with Cdt1, and it enhances Cdt1-dependent rereplication. Thus, HBO1 plays a direct role at replication origins as a coactivator of the Cdt1 licensing factor. Since HBO1 is also a transcriptional coactivator, it has the potential to integrate internal and external stimuli to coordinate transcriptional responses with initiation of DNA replication (Miotto, 2008).

The proteins comprising the pre-RC (ORC and MCM complexes, Cdt1, Cdc6) are conserved from yeast to human, and regulated proteolysis of Cdt1 is critical for licensing replication origins such that they fire only once per cell cycle. Metazoans have additional mechanisms to control pre-RC formation such as inhibition of Cdt1 licensing activity by Geminin. Intriguingly, HBO1 does not appear to exist in yeasts or worms, suggesting that HBO1 is a recently evolved molecule in the licensing process and indicating species-specific differences in the mechanism of MCM complex loading. In yeast, a direct interaction with Cdt1 is critical for loading the MCM complex, whereas in human cells the Cdt1-recruited HBO1 is critical. It is unknown to what extent direct interactions between Cdt1 and the MCM complex are important for recruitment of the MCM complex to origins in human cells. However, the fact that Geminin inhibits Cdt1-dependent licensing, but not HBO1 association, suggests a role for a direct Cdt1-MCM complex interaction (Miotto, 2008).

As might be expected for a recently evolved function, HBO1 appears to affect different steps in DNA replication in a species-specific manner. As shown in this study for human cells, HBO1 affects Cdt1 licensing activity but not its chromatin loading. However, in Drosophila, targeting of Chameau (the HBO1 homolog) in the vicinity of an artificial chorion origin promotes ORC redistribution on origins and replication initiation, whereas depletion of HBO1 in Xenopus extracts affects Cdt1 association with chromatin but not ORC recruitment. It is noted, however, that these apparent species-specific differences in the molecular function of HBO1 may reflect the assays used (Miotto, 2008). .

HBO1 also functions as a transcriptional coactivator for hormone receptors and AP-1 proteins, and it is speculated that it may have evolved from this role to become a coactivator for the Cdt1 licensing factor. In addition, the dual role of HBO1 as a coactivator for transcriptional regulation and DNA licensing suggest the possibility that HBO1 might integrate internal and external stimuli to coordinate transcriptional responses with initiation of DNA replication. In particular, DNA damage activated p53 transcription factor regulates HBO1 enzymatic activity and may regulate HBO1 function during DNA licensing (Miotto, 2008).

Transcriptional regulation of Cdt1

Geminin and Cdt1 play an essential role in the initiation of DNA replication, by regulating the chromatin loading of the MCM complex. The transcription of human Geminin and Cdt1, as well as that of MCM7, is activated by transcription factors E2F1-4, but not by factors E2F5-7. Analysis of various Geminin and Cdt1 promoter constructs shows that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the transcriptional activation. The promoter activity for human Geminin was activated by the E7, but not E6, oncogene of human papillomavirus type 16. While E2F1-induced activation of human Cdt1 gene transcription was suppressed by pRb, but not by p107 or p130, its E2F4-induced activation was suppressed by pRb, p107, and p130. Furthermore, the promoter activities of human Geminin and Cdt1 were demonstrated to be growth-dependent. Taken together, the results demonstrate that Geminin and Cdt1 constitute targets for various members of the E2F family of transcription factors, and that expression of Geminin and Cdt1 is perhaps mediated by the activation of a pRb/E2F pathway (Yoshida, 2004).

Cdt1 phosphorylation by cyclin dependent kinases and Cdt1 degradation

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. The CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. It is proposed that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1 (Zhong, 2003).

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. Radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. This study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage (Higa, 2003).

DNA replication initiation is tightly controlled so that each origin only fires once per cell cycle. Cell cycle-dependent Cdt1 degradation plays an essential role in DNA replication control; overexpression of Cdt1 leads to re-replication. In this study, the mechanisms of Cdt1 degradation were investigated in mammalian cells. The F-box protein Skp2 specifically interacts with human Cdt1 in a phosphorylation-dependent manner. The SCF(Skp2) complex ubiquitinates Cdt1 both in vivo and in vitro. Down-regulation of Skp2 or disruption of the interaction between Cdt1 and Skp2 results in a stabilization and accumulation of Cdt1. These results suggest that the SCF(Skp2)-mediated ubiquitination pathway may play an important role in the cell cycle-dependent Cdt1 degradation in mammalian cells (Li, 2003 ).

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. With SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically express DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevents UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. It is suggested that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation (Hu, 2004).

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, it was found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 is caffeine-insensitive, the UV-triggered degradation of Cdt1 is caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibits the degradation of Cdt1 and results in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induces the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered with the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage (Kondo, 2004).

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. The SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. Human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells (Liu, 2004).

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines it has been demonstrated that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. In contrast, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. The data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication (Nishitani, 2004).

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, human Cdt1 is shown to be phosphorylated by cyclin A-dependent kinases dependent on Cdt's cyclin-binding motif. Cdk phosphorylation results in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 is inhibited by the phosphorylation. However, geminin binding to Cdt1 is not affected by the phosphorylation. Finally evidence is provided 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 (Sugimoto, 2004).

The N terminus of Cdt1 is required for its degradation during S phase. The stabilization has been attributed to deletion of the cyclin binding motif (Cy motif), which is required for Cdt1 phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, it was found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. This study has localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delays entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase (Takeda, 2005).

Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and by Geminin is essential to prevent re-replication. The N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. These data unravel multiple distinct pathways regulating Cdt1 to block re-replication (Nishitani, 2006).

PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication

Ubiquitin-mediated proteolysis of the replication licensing factor Cdt1 (Cdc10-dependent transcript 1) in S phase is a key mechanism that limits DNA replication to a single round per cell cycle in metazoans. In Xenopus egg extracts, Cdt1 is destroyed on chromatin during DNA replication. Replication-dependent proteolysis of Cdt1 requires its interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric processivity factor for DNA polymerases. Cdt1 binds to PCNA through a consensus PCNA-interaction motif that is conserved in Cdt1 of all metazoans, and removal of PCNA from egg extracts inhibits replication-dependent Cdt1 destruction. Mutation of the PCNA-interaction motif yields a stabilized Cdt1 protein that induces re-replication. DDB1, a component of the Cul4 E3 ubiquitin ligase that mediates human Cdt1 proteolysis in response to DNA damage, is also required for replication-dependent Cdt1 destruction. Cdt1 and DDB1 interact in extracts, and DDB1 chromatin loading is dependent on the binding of Cdt1 to PCNA, which indicates that PCNA docking activates the pre-formed Cdt1-Cul4(DDB1) ligase complex. Thus, PCNA functions as a platform for Cdt1 destruction, ensuring efficient and temporally restricted inactivation of a key cell-cycle regulator (Arias, 2006).

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. The N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage (Senga. 2006).

Cdt1, cell cycle and cancer

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 predisposes cells for malignant transformation. Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. The data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. These data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells (Xouri, 2004).

double parked: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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