Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress

The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. Destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation is not sufficient to explain the rapid destruction of Dup at G1-S. Evidence is presented that it is DNA replication itself that triggers rapid Dup destruction. A range of defects in DNA replication stabilize Dup protein and this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress is cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, it was found that cells with high levels of Dup also have elevated levels of Gem protein. It is proposed that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails (May, 2005).

Functional Characterization and Protein Interactions

To confirm that the Drosophila Geminin-related protein was in fact the Drosophila homolog of Geminin, its effect on DNA replication was determined using a cell-free replication extract from unfertilized Xenopus eggs. In this system, the template for replication is Xenopus sperm head DNA and the reaction is started by calcium addition that mimics fertilization. Addition of bacterially produced Drosophila Geminin completely inhibits the incorporation of [alpha-32P]dCTP into DNA at concentrations of 1-4 µg/mL (50-150 nM). The same concentration of Xl Geminin was required to inhibit replication in the extract (Quinn, 2001).

To determine whether Drosophila Geminin would inhibit binding of Mcms onto the pre-RC, chromatin was pelleted from the replication assays and the amount of Mcm complex bound to the chromatin was determined by immunoblotting. Drosophila Geminin inhibits Mcm binding at concentrations >2 µg/mL, which also inhibits DNA replication. The protein has no effect on the binding of Cdc6 or Orc complex to chromatin. It is concluded that the Drosophila Geminin, like Xl Geminin, inhibits DNA replication by preventing Mcm binding to chromatin (Quinn, 2001).

Recent studies have shown that Geminin acts to inhibit DNA replication by binding to and preventing Cdt1 from loading Mcms onto the pre-RC complex (Wohlschegel, 2000; Tada, 2001). Whether Drosophila Geminin could form a complex with Drosophila Cdt1 homolog Doubleparked (Dup) was investigated in vivo. Western analysis of Drosophila embryonic extracts using antisera to the Drosophila Geminin specifically detects two bands at 25 kD and 30 kD (slightly larger than the predicted size of ~22 kD), the abundances of which are increased in extracts after heat shock-induced expression of Drosophila Geminin and are decreased in extracts made from embryos from l(2)k14019 or l(2)k03202 mutant flies, where one-half of the wild-type level is expected. It is concluded from these data that both bands are Drosophila Geminin (Quinn, 2001).

Immunoprecipitation-Western analysis of embryonic extracts using antibodies to Geminin and to Dup reveal that these proteins form a complex in vivo. When the Geminin-related antibody is used in the immunoprecipitation, Dup (which gives three bands >75 kD) is coprecipitated. In the converse experiment, immunoprecipitation with the Dup antibody coprecipitates the 30-kD Geminin but not the 25-kD band. It is concluded that the 30-kD Drosophila Geminin forms a complex with Dup in vivo. Therefore, the Drosophila Geminin behaves similarly to Xl and Hs Geminin by two different criteria --the inhibition of DNA replication by preventing the loading of Mcms and complex formation with Cdt1(Dup) (Quinn, 2001).

Cell type-dependent requirement for PIP box-regulated Cdt1 destruction during S phase

DNA synthesis-coupled proteolysis of the prereplicative complex component Cdt1 by the CRL4(Cdt2) E3 ubiquitin ligase is thought to help prevent rereplication of the genome during S phase. To directly test whether CRL4(Cdt2)-triggered destruction of Cdt1 is required for normal cell cycle progression in vivo, a mutant version was expressed of Drosophila Cdt1 (double parked; dup), which lacks the PCNA-binding PIP box (DupδPIP) and which cannot be regulated by CRL4(Cdt2). DupδPIP is inappropriately stabilized during S phase and causes developmental defects when ectopically expressed. DupδPIP restores DNA synthesis to dup null mutant embryonic epidermal cells, but S phase is abnormal, and these cells do not progress into mitosis. In contrast, DupδPIP accumulation during S phase did not adversely affect progression through follicle cell endocycles in the ovary. In this tissue the combination of DupδPIP expression and a 50% reduction in Geminin gene dose resulted in egg chamber degeneration. No Dup hyperaccumulation was detected using mutations in the CRL4(Cdt2) components Cul4 and Ddb1 (piccolo), likely because these cause pleiotropic effects that block cell proliferation. These data indicate that PIP box-mediated destruction of Dup is necessary for the cell division cycle and suggest that Geminin inhibition can restrain DupδPIP activity in some endocycling cell types (Lee, 2010).

Accurate genome duplication during cell cycle progression requires assembly of a prereplicative complex (pre-RC) at origins of DNA replication. Pre-RCs contain the origin recognition complex (ORC), Cdc6, and Cdc10-dependent transcript1 (Cdt1) proteins, which assemble at origins during late mitosis/G1 and recruit the minichromosome maintenance complex (MCM2–7), a core component of the replicative DNA helicase. After DNA synthesis is initiated, pre-RC components are displaced from the chromatin and prevented from reassembling until the next G1 via multiple mechanisms including nuclear export, inhibitory phosphorylation, and ubiquitin-mediated proteolysis (Lee, 2010).

Preventing pre-RC assembly and reloading of the MCM complex within S phase is crucial to prevent rereplication, which can cause DNA damage and genomic instability that may contribute to cancer. Negative regulation of Cdt1 is a key aspect of pre-RC assembly in metazoans, as increased Cdt1 activity is sufficient to trigger rereplication in many situations. Moreover, recent experiments in mice suggest that Cdt1 overexpression may promote tumor formation or progression. Metazoan Cdt1 activity is negatively regulated by two mechanisms: regulated proteolysis and binding to the protein Geminin. Geminin blocks the ability of Cdt1 to load the replicative helicase at origins, most likely because the Geminin and MCM2–7 binding domains of Cdt1 overlap. Studies in mammalian and Drosophila cells have shown that the loss of Geminin function can cause rereplication, indicating that this inhibitory mechanism is required for normal genome duplication in some cell types (Lee, 2010).

After origins are licensed, Cdt1 is rapidly destroyed upon the onset of DNA replication via ubiquitin-mediated proteolysis. Cdt1 proteolysis is controlled by two members of the Cullin-RING family of E3 ubiquitin ligases (CRL): CRL1 (aka SCF) and CRL4. These two ligases utilize different mechanisms for targeting Cdt1. Phosphorylation of Cdt1 by S phase cyclin-dependent kinases (e.g., cyclin E/Cdk2) is mediated by a conserved cyclin binding (Cy) motif and triggers ubiquitylation by CRL1Skp2. CRL4Cdt2 directs replication-coupled destruction of Cdt1 through a degron at the Cdt1 NH2-terminus containing a motif called a PIP (PCNA-interacting polypeptide) box. The PIP box confers direct binding to PCNA at replication forks after the initiation of S phase, and the PIP box–containing degron recruits CRL4Cdt2 for ubiquitylation and subsequent destruction of Cdt1. In human cells these pathways act redundantly, as mutations in both the PIP box and Cy domains are necessary to stabilize Cdt1 in S phase. In other situations there appears to be no redundancy between these ligases. For instance, Cul4 loss of function in Caenorhabditis elegans causes Cdt1 hyperaccumulation and rereplication. Cdt1 is also destroyed after DNA damage, and CRL4 depletion or mutations in the PIP box block this destruction in fission yeast, Drosophila, and mammalian cells (Lee, 2010).

The degree of redundancy or cell-type specificity between CRL- and Geminin-mediated inhibition of Cdt1 during animal development is not completely understood. For instance, if Geminin is sufficient for Cdt1 regulation in all cell types, cell cycle progression should not be affected when Cdt1 destruction is inhibited. To test the significance of Cdt1 destruction during development, the Drosophila homolog of Cdt1, double parked (Dup) was studied. Dup is required to initiate DNA replication and is degraded promptly upon S phase entry. Dup contains a Cy domain that is important for its normal function and mediates regulation by cyclin E/Cdk2 as well as a conserved PIP box whose function has yet to be specifically studied (Lee, 2010).

Although many previous studies have focused on the molecular mechanisms of Cdt1 regulation, they have not directly addressed whether loss of CRL4Cdt2 regulation of Cdt1 disrupts cell cycle progression in vivo. Advantage was taken of the well-characterized dup null mutant phenotype to test whether a mutant version of Dup protein lacking the PIP box could provide normal function in the absence of endogenous Dup. The results indicate that PIP box-dependent regulation is necessary for rapid Dup destruction during S phase and for normal progression of the embryonic cell division cycle, but not for normal endocycle progression in a cell type where Gem function can compensate for Dup stabilization in S phase. Thus, specific cell types depend on different modes of Cdt1 regulation during normal animal development (Lee, 2010).

The results indicate that deletion of the PIP box prevents the rapid destruction of Dup at the beginning of S phase. Before discovery of the PIP degron/CRL4 mechanism of replication-coupled proteolysis, a similar result was reported with a mutant version of Dup lacking the NH2-terminal 46% of the protein, including the PIP box. Thus, the current results suggest that the previous observation is due to deletion of the PIP degron. Biochemical and genetic experiments from a number of species suggest that the PIP degron recruits proteins to chromatin-bound PCNA at replication forks during S phase. These proteins are subsequently ubiquitylated by CRL4Cdt2 and proteolyzed. Although this study did not detect hyperaccumulation of Dup in imaginal cells mutant for components of CRL4Cdt2, the PIP degron mechanism is conserved in Drosophila, and CRL4Cdt2 is required for Dup destruction after DNA damage in cultured S2 cells. Ohenotypic pleiotropy resulting from abrogation of CRL4Cdt2 function may have masked the ability to detect effects on Dup protein (Lee, 2010).

Interestingly, deletion of the PIP box resulted in inappropriate Dup accumulation in only about half of BrdU-positive S phase cells. CRL1 and CRL4 act redundantly in triggering human Cdt1 destruction during S phase. In contrast, the current results suggest that cyclin E/Cdk2-dependent phosphorylation and CRL1 ubiquitylation of Cdt1 do not contribute significantly to Dup destruction during S phase and thus likely do not account for the disappearance of DupδPIP from BrdU-positive cells. One recently proposed possibility is that CRL1-dependent regulation of Cdt1 arose in higher metazoans (Lee, 2010).

By using the rescue of dup embryonic mutant phenotypes as an assay, the data clearly demonstrate that DupδPIP is unable to support progression through the cell division cycle. Similarly, DupδPIP expression in WT embryos caused cell cycle arrest in interphase. In these experiments there was no obvious large increase in DNA content, as occurs from rereplication in other cell types after overexpression of Cdt1 or depletion of Cdt1 regulatory mechanisms (e.g., CRL4 or Gem). Also no extensive DNA damage or apoptosis was detected. It is proposed that the near physiological levels of DupδPIP expression achieved in these experiments, as suggested by the ability to phenotypically rescue dup mutant cells using transgenic WT Dup, causes a small number of replication origins to reinitiate. This situation results in a low level of DNA damage that activates a checkpoint and arrests cells in interphase. Alternatively, DupδPIP may block DNA synthesis more directly, as a recent study reported that excess Cdt1 prevents nascent DNA strand elongation (Lee, 2010).

Previous studies reported that heat-shock driven overexpression of Dup in endocycling follicle cells cause rereplication, and that Cul4 mutant follicle cells hyperaccumulate Dup and exhibit replication defects during gene amplification (Lin, 2009). This study found that Gal4-driven expression of DupδPIP did not cause either of these phenotypes and did not dramatically alter endocycle S phase or chorion gene amplification. As in the embryo, it is proposed that the lack of large increases in DNA content seen in the experiments with DupδPIP is due to lower expression levels of Dup than that obtained by Thomer (2004). Also, a small amount of DNA damage might not disrupt the endocycle. Lin (2009) showed that ectopic genomic BrdU incorporation during gene amplification stages occurs in Cul4 or Ddb1 mutant follicle cells. The same phenotype was not observed after DupδPIP expression, suggesting that these replication defects may be due to misregulation of another CRL4 target (Lee, 2010).

Several observations suggest the possibility that Cdt1 is regulated in a cell-type specific manner. In Drosophila S2 cells and mammalian cells, RNAi against Gem but not Cul1 or Cul4 results in rereplication. In contrast, Drosophila Gem is not required for proliferation of imaginal discs or endoreplication in salivary glands. Null mutations of C. elegans Cul4 or Ddb1 cause overreplication primarily in seam cells. Finally, ectopic expression of Arabidopsis Cdt1 induced overreplication only in endocycling cells. The basis for these cell type differences is not known (Lee, 2010).

This study showed that reduction of Gem gene dose in combination with DupδPIP expression in follicle cells causes deterioration of egg chambers during oogenesis. The possibility is favored that Dup inhibition by Gem can compensate for the loss of PIP-mediated destruction of Dup in this cell type. In proliferating embryonic ectodermal cells, loss of PIP-mediated Dup destruction was sufficient to block the cell cycle, suggesting that Gem activity is unable to provide compensatory inhibition of Dup in this situation. Cell type specific differences in Gem expression or activity could explain why cells are differently sensitive to stabilized Dup. For instance, the C. elegans Gem homolog, GMN-1, is expressed at higher levels in the germ line, suggesting that this tissue might be buffered against disruption of Dup destruction as was observed in Drosophila follicle cells. In some cell types Gem levels increase concomitantly with increased levels of Dup after DNA replication is compromised. Determining the mechanisms by which certain cell types are more sensitive to mis-regulation of Cdt1 destruction than others will be necessary for a complete understanding of replication control in developing organisms (Lee, 2010).

Knockdown of SCFSkp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes

In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, data was considered from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like (Cul-1), SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCFSkp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type (Kroeger, 2013).

The generation of an eaterGal4; UAS-GFP strain allowed identification the functional importance of SCF complex members for the plasmatocyte blood cell lineage by a RNAi knockdown approach. Using this technique, several genes belonging to the core SCF complex were identified that, when knocked-down, caused a very distinctive giant cell phenotype. Importantly, as eater was bing used as a driver to identify complex components, it was confirmed that these enlarged cells were plasmatocytes by anti-P1 plasmatocyte-specific antibody staining. This suggested, as proof-of-principle, that knockdown of gene function in mature plasmatocytes could elicit aberrant phenotypes dependent on the functional requirement of an essential gene/gene complex (Kroeger, 2013).

Previous research has shown that there are several Drosophila genes that may be involved in SCF complexes in order to determine specificity for a substrate. The F-box is thought to convey specificity of this complex by recruiting the substrate, however activation of the Cullin by neddylation factors also plays a role in ubiquitation of the substrate. A comprehensive list of all known and predicted complex members was used to identify the remaining members of the specific SCF complex that function in Drosophila hematopoiesis, as knockdown of only one of each of the core components caused enlarged plasmatocytes. lin19, SkpA and Roc1a likewise play a role in the hematopoietic niche, the PSC of the larval lymph gland. Knockdown of these genes caused a decrease in the number of PSC cells, as well as an increase in the size of these cells. These data, along with the findings in this current study, suggest that the SCF complex has a significant role in multiple aspects of Drosophila larval hematopoiesis (Kroeger, 2013).

Using fluorescence microscopy, it was noted that the enlarged cells caused by the SCF knockdown had a significant excess of DNA in the nuclear region. To investigate the hypothesis that DNA re-replication was occurring in plasmatocytes with the SCF complex knockdown, anti-gamma- Tubulin staining of centrioles was performed. Previously, it was shown that knockdown of Gem elicits DNA re-replication, therefore this study used it as a positive control. It was evident that the lin19 knockdown had multiple centrioles in one giant plasmatocyte, similar to plasmatocytes from the gem RNAi samples. It was also clear that the DNA had replicated many times, without any cellular division as indicated by BrdU-positive, but phospho-Histone H3-negative enlarged cells. These data support the idea that plasmatocytes from SCF knockdown animals undergo DNA re-replication, thus the SCF complex is necessary for Dup degradation. Additionally, previous research had identified a number of proteins that when misexpressed or knocked-down cause an enlarged cell phenotype with excess DNA replication. Several papers have shown that misregulation of Cyclin E can cause aberrant DNA synthesis. Research has also suggested that knockdown of Gem can cause this excessive DNA phenotype. In the current experiments, antibody staining identified that the subcellular localization of both these proteins changed between control samples and the lin19 knockdown. Importantly, Dup is necessary for DNA replication, but it must be degraded to prevent re-replication. As the main role of Gem is to inhibit Dup, and Gem was no longer found in the nucleus in the knockdown, this is suggestive that Gem had complexed with Dup, removing it from the nucleus. Conversely, Cyclin E was found in the nucleus. This is notable because Cyclin E is known to phosphorylate Dup marking it for ubiquitination, leading to its nuclear localization. It is also known that SCFSkp2 degrades Cyclin E. This is another explanation for the accumulation of Cyclin E in the nucleus of SCF knockdown hemolymph samples. These data suggest that Dup may be the target substrate for the SCF complex being studied, with a secondary target possibly being Cyclin E. Previous research in human cells has shown that SCFSkp2 regulates the degradation of Cdt1 (the homolog of Drosophila Dup)(Li, 2003). It has also been shown that the activated SCFSkp2 complex plays a role in murine hematopoiesis, by ubiquitinating proteins necessary for proper cell cycle, such as Cyclin E. There are still many questions to be answered about SCF regulation in blood cells, as some of these results are contradictory (Kroeger, 2013).

In addition to these data, protein localization in the knockdown of Cyclin E showed that Gem had been removed from the nucleus, again consistent with the notion that it was titrated away from the nucleus by binding Dup. This is plausible because the SCF complex can recognize its substrates due to phosphorylation state. Since Cyclin E was knocked-down, Dup was not properly phosphorylated, and it was not recognized as the substrate by the SCF complex, therefore never being ubiquitinated nor degraded. Furthermore, in the Cyclin E knockdown, Dup localized to the nucleus similar to its localization in the SCF knockdown. This would make it necessary for Gem to inhibit Dup, causing Gem to take on a non-nuclear localization, while Dup would have a nuclear localization, if Dup was in excess. Taken together, these lines of investigation support the hypothesis that Cyclin E is necessary to phosphorylate Dup, allowing the SCF complex to recognize and ubiquitinate it. Dup that remains in the nucleus after degradation must be bound by Gem for the cell cycle to progress properly. DNA re-replication will occur if Dup remains in the nucleus. It is highly suggestive that knockdown of Cyclin E or the SCF complex perturbs this mechanism, causing Dup accumulation in the nucleus, and the cells to re-initiate DNA replication. Furthermore, others have shown there must be a balance of Gem and Dup in the nucleus for proper progression through the cell cycle. This research shows that there is a lack of Gem and an accumulation of Dup in the nucleus, which leads to excessive DNA replication and additional centriole replication in five percent of the plasmatocyte population (Kroeger, 2013).

Although re-replication is one mechanism to explain the SCF loss-of-function phenotype, a similar non-canonical process, known as endoreplication, could also account for the over-replicative system in these cells. Endoreplication is a cycle in which cells undergo S phases that are separated only by gap phases but not an intervening mitosis. However, endoreplication is not known to occur in wild-type Drosophila plasmatocytes. Further, Drosophila plasmatocytes are most similar to mammalian macrophages, which also do not endoreplicate. Since several of the proteins studied in this paper have been implicated in re-replication with phenotypes including enlarged cells, increased DNA content, and multiple centriole replication, the hypothesis is favored that re-replication is triggered in plasmatocyte development in the absence of SCF complex activity (Kroeger, 2013).

It is intriguing that only five percent of the cells display the re-replication phenotype. One explanation is that the smaller cells have arrested. There are many intrinsic mechanisms to ensure proper cell cycle progression preventing re-replication and ultimately cancer. It is possible these enlarged cells have escaped these mechanisms, causing the cell to replicate their DNA many times without going through mitosis, while the smaller cells arrest, to prevent this phenotype. It is also possible that only five percent of these cells are going through cell division during the time the RNAi is functionally knocking down the gene. Previous research has suggested that during mid-to-late third instar larval stages, only one to two percent of cells are going through mitosis at a given time. eaterGal4 is activated during second instar, however there is likely a latent period between activation of Gal4 and protein knockdown by the RNAi. This is consistent with only five percent of cells having an active cell cycle, and becoming enlarged through re-replication. A final possibility is that there are partially redundant mechanisms for the regulation of Dup. As previously described, the SCF complex has been shown to be involved in the ubiquitination and subsequent degradation of Dup, and Gem will inhibit the remainder of the Dup that may be in the nucleus. There may be additional mechanisms which ubiquitinate or inhibit Dup, therefore avoiding re-replication. The smaller cells may have activated one of these mechanisms to aid the cell in proper cell cycle, ultimately avoiding cancer. The regulation of Dup is of vast importance, and there are several possibilities of alternate mechanisms to prevent the re-replication phenotype elicited by cells which have excess Dup in the nucleus (Kroeger, 2013).

To further implicate the necessity of Dup regulation in the proper cell cycle of plasmatocytes, a rescue experiment was performed by overexpressing the Dup inhibitor, Gem. By overexpressing this inhibitory protein, it was hypothesized that the nuclear localization of Gem would increase, the protein would bind Dup, and therefore decrease the re-replication that is observed in SCF complex knockdown. Performing immunohistochemistry experiments identified that there was an increase in nuclear Gem and a decrease in Dup. Additional experimental evidence supports this hypothesis as there is a decrease in size of plasmatocytes with genotype pxnGal4>UAS-Gem43>UAS-lin19 RNAiHM05197 compared with pxnGal4>UAS-lin19 RNAiHM05197. There is a drastic decrease in the number of giant cells, which are larger than 25.1 μm, in pxn>UAS-Gem43>UAS-lin19 RNAiHM05197 (8/100) plasmatocytes compared with SCF knockdown hemocytes (45/100). It was also noted that there was a significant decrease in the average size of plasmatocytes in hemolymph samples from Gem overexpression in the SCF knockdown background (p<0.001). These lines of evidence are all suggestive that knockdown of the SCF complex increased nuclear Dup leading to re-replication. By over-expressing its inhibitor, Gem, it is possible to partially rescue this enlarged cell phenotype generated by excess nuclear Dup. These data suggest the regulation of Dup is important in the proper cell cycle progression of plasmatocytes. Furthermore, these data support the hypothesis that the SCFSkp2 complex is responsible for the ubiquitination of Dup, allowing plasmatocytes to proliferate properly. Although this study provides substantial genetic evidence that the SCFSkp2 complex is necessary to ubiquitinate Dup allowing for proper hematopoietic cell cycle progression, future studies using biochemical techniques to show physical interactions are needed to support the model proposed here (Kroeger, 2013).

Furthermore, there are two ubiquitin ligase complexes known to be involved in the ultimate degradation of Dup: The SCFSkp2 complex, described in this manuscript, and the Cul4-DDB1-CDT2-PCNA (Cul4CDT2) complex. To vastly decrease the possibility that the Cul4CDT2 complex was responsible for the enlarged cell phenotype, both DDB1 and PCNA were knocked-down via RNAi and Cul4 mutants were also analyzed. Although DDB1 functional knockdown elicited a small number of enlarged cells, these cells had a different morphology than the SCFSkp2 knockdown. Additionally, none of the other analyses elicited any enlarged cells as observed when the SCFSkp2 complex was knocked-down. This further implicates the necessity of the SCFSkp2 complex in the proper plasmatocyte cell cycle (Kroeger, 2013).

To summarize, this manuscript identifies the SCF ubiquitin ligase complex as a novel regulator of plasmatocytes. Genetic evidence is presented that suggests that Dup is the main target for the SCFSkp2 complex. It is proposed that the SCFSkp2 complex plays an integral role in Drosophila hematopoiesis by ubiquitinating Dup, which is necessary for proper cell cycle progression. Knockdown of the SCF complex causes an accumulation of Dup in the nucleus, inducing the cell to undergo multiple rounds of replication without an intervening mitosis or cytokinesis. This causes some plasmatocytes to become vastly enlarged, with multiple centrioles and excessive DNA content. Taken together, these findings provide evidence that the SCF complex is necessary for proper cell cycle progression during plasmatocyte development in Drosophila. As the SCF complex is conserved from Drosophila to humans, these findings implicate the importance of the roles of ubiquitin ligase complexes in the cell cycle and their potential malfunctions in blood cell cancers (Kroeger, 2013).



To explore the distribution of Geminin during Drosophila development, embryos and larval tissue were stained with the anti-Geminin antibody. To demonstrate that the antibody specifically recognizes Geminin in vivo, embryos ectopically expressing geminin under control of the engrailed (en) driver were examined. En is expressed in stripes and Geminin protein is dramatically elevated in stripes coincident with the En pattern. In contrast, homozygous Geminin mutant embryos showed considerably reduced staining with anti-Geminin antibody, compared with wild type. Taken together, these results demonstrate that the anti-Geminin antibody is specific for Geminin in vivo (Quinn, 2001).

Staining of Drosophila embryos with the anti-Geminin antibody and with propidium iodide to stain DNA shows that Geminin is located to the nucleus. Therefore, like Xl Geminin (McGarry, 1998), Geminin is a nuclear localized protein. Throughout embryogenesis, Geminin protein expression is correlated with mitotically dividing or endoreplicating cells. Early in embryogenesis during the rapid syncitial divisions, Geminin is present at high levels irrespective of cell cycle stage. In the G2 regulated cycles 14-16, Geminin is present in a dynamic pattern similar to the domains of mitosis that occur at this stage. At the stage where cells of the peripheral nervous system (PNS) and central nervous system (CNS) divide, Geminin is absent from the G1-arrested cells of the epidermis but present in the dividing neural cells. Later in development, Geminin is absent in the PNS cells, which have stopped dividing, but present in the dividing CNS cells. Geminin persists in the pole (germ) cells that are arrested in G2 at this stage. In addition, Geminin is detected in the endoreplicating tissues of the gut (Quinn, 2001).

The dynamic pattern of Geminin expression during embryogenesis is similar to that of Cyclin A or B, which accumulate during late S-G2 and are degraded during mitosis. To explore the cell cycle distribution of Geminin further, anti-Geminin staining was examined relative to cell cycle markers of S phase (BrdU-labeling), mitosis (anti-PH3 antibody staining), and late S phase until the metaphase to anaphase transition (anti-Cyclin B staining). Geminin and BrdU-labeled cells show partial overlap. Small, weakly BrdU-labeled cells (early S phase) do not contain Geminin; strong BrdU-labeled cells show higher levels of Geminin; and large non-BrdU-labeled cells (G2/M) show the highest levels of Geminin, consistent with Geminin accumulating during S-G2 phase. Co-localization of Geminin with PH3 shows that as cells enter metaphase and DNA is stained strongly with anti-PH3, Geminin is still present but reduced in level. In anaphase, when anti-PH3-stained chromosomes separate, Geminin is undetectable. Geminin protein distribution during the cell cycle is remarkably similar to Cyclin B although Geminin is nuclear-localized and Cyclin B is cytoplasmic. Some small interphase cells (likely to be in early S phase) were observed to contain Geminin but not Cyclin B, suggesting that Geminin begins to accumulate before Cyclin B. Taken together, these data show that Geminin, like Xl Geminin and Drosophila Cyclin B, accumulates during S phase and is degraded at the metaphase to anaphase transition (Quinn, 2001).


In the eye imaginal disc, cell proliferation occurs in a spatial arrangement. Geminin is present in the region of asynchronous dividing cells in the anterior region and in a band posterior to the morphogenetic furrow (MF), where the synchronous S phases occur, but not in the G1-arrested cells within the MF. Geminin shows only partial overlap with cells staining with the mitotic marker anti-phospho histone H3 (PH3) that stains DNA in all stages of mitosis. In the posterior part of the eye imaginal disc where many cells are differentiating, Geminin is present in a subset of cells that may represent the undifferentiated G2-arrested cells that are present in this region. These results show that Geminin is present in proliferating cells and is at high levels in S-G2 phase cells, at low levels or absent in some mitotic cells, and absent from G1 cells (Quinn, 2001).


Geminin is also expressed during adult ovary development where endoreplication occurs in the nurse cells within the egg chamber and in the surrounding follicle cells. The follicle cells undergo genomic endoreplication until stage 10A and switch to DNA replication amplification of specific foci including the chorion genes at stage 10B. Early in oogenesis Geminin is present in the nuclei of the endoreplicating nurse cells, but at later stages Geminin is present in the nuclei and cytoplasm. The redistribution of Geminin to the cytoplasm correlates with nurse-cell apoptosis before dumping of the nurse cell cytoplasm into the oocyte. Geminin is present in the nuclei of the follicle cells throughout oogenesis and is not specifically localized to the amplification foci at stage 10B, as occurs with replication proteins Dup and Orc2. In stage-12 egg chambers, when amplification becomes limited to the anterior follicle cells, Geminin protein is observed in the nuclei of all follicle cells and remains in all follicle cells until the end of oogenesis. Therefore, Geminin is present in mitotic and endoreplicating cells at different stages of development, consistent with a role in all replicative cycles throughout development (Quinn, 2001).

Effects of Mutation and Overexpression

To determine whether overexpression of Geminin acts to inhibit DNA replication in vivo, transgenic flies were generated that contain geminin under control of the S. cerevisiae UAS(GAL4) promoter. Ectopic overexpression of Geminin during embryogenesis by heat shock induction of hsp70-GAL4 UAS-geminin flies results in a general decrease in BrdU-labeling cells in mitotic and endoreplicating tissues. To demonstrate this effect more clearly, the en-GAL4 driver was used to overexpress Geminin in a striped pattern during embryogenesis. Ectopic overexpression of Geminin results in a dramatic decrease in S-phase cells within the En stripe, relative to surrounding cells. Propidium iodide staining of En-Geminin-expressing cells reveals more condensed nuclei within the En stripes, suggesting that cells were attempting to enter mitosis. Staining with anti-PH3 to detect mitotic cells showsthat many cells (4× as many as normal) in the En-Geminin stripe are in mitosis. A similar phenotype is observed in Dup mutants despite the fact that they fail to replicate their DNA (Whittaker, 2000) and occurs presumably because the DNA replication checkpoint can only be triggered after the loading of DNA polymerase alpha onto the pre-RC. To determine the fate of these cells, TUNEL was carried out to detect apoptotic cells. Wild-type embryos at stage 11 normally show very little TUNEL staining, whereas the En-Geminin stage-11 embryos show numerous TUNEL-positive cells associated with the En stripes. These data show that ectopic overexpression of Geminin results in inhibition in DNA replication, increased numbers of metaphase cells, and increased apoptosis (Quinn, 2001).

Ectopic overexpression of Geminin using the eyeless(ey)-GAL4 driver, which is expressed during the early proliferative phase of the eye-antennal imaginal disc, also results in a dramatic decrease in S phases and in the size of third instar larvae eye discs and the size of the adult eye. Overexpression of Geminin using the GMR-GAL4 driver, which is expressed posterior to the MF in the eye imaginal discs of third instar larvae, leads to a 40%-50% decrease in S-phase cells within this region, but to severely rough adult eyes. Taken together, these data show that ectopic overexpression of Geminin leads to an inhibition of S phases in both mitotic and endoreplicative cycles and at different developmental stages (Quinn, 2001).

The GMR-geminin rough eye phenotype represents a good phenotype in which to examine genetic interactions. This phenotype is responsive to the dose of geminin because two copies of UAS-geminin results in a less severe phenotype compared with three copies of the transgene. In addition, reducing the dose of endogenous geminin by half using the strong P alleles results in a less severe phenotype. To determine whether geminin genetically interacts with dup(cdt1), the dosage of dup was reduced by half using a null allele (dupa1; Whittaker, 2000) in a GMR-GAL4 UAS-geminin (two copies) background. Halving the dosage of dup enhances the GMR-geminin eye phenotype, leading to a smaller, rougher eye. Moreover, the dupa1 mutant embryonic cycle 16 S-phase defect is suppressed by a geminin mutant. Therefore, consistent with the biochemical interaction observed between Geminin and Dup, geminin genetically interacts with dup (Quinn, 2001).

Of the three P element alleles that are inserted within or 5' of the geminin transcriptional unit, l(2)09107 is a partially female sterile allele, whereas l(2)k14019 and l(2)k03202 are embryonic/larval lethal. The P element allele l(2)k14019 mutant phenotype is a third instar larval lethal with reduced imaginal discs. When crossed to a deficiency of the region [Df(2R)ST1], 40% of l(2)k14019/Df and l(2)k03202/Df embryos died before hatching and the rest died during larval development. Occasional l(2)k14019/Df trans-heterozygous third instar larvae were observed but no third instar l(2)k03202/Df larvae were detected, suggesting that l(2)k03202 is a stronger allele than l(2)k14019 (Quinn, 2001).

Because l(2)k03202 is the strongest allele, focus was placed on examining l(2)k03202/Df(2R)ST1 trans-heterozygous embryos for cell cycle defects. During early embryogenesis, the overall S-phase patterns of the mutant appear normal and cells exit from S phase at the appropriate developmental time. To examine mitoses during cycle 14-16, mutant embryos were stained using anti-PH3 to detect mitotic chromosomes and anti-Actin to visualize cell outlines. Interestingly, l(2)k03202/Df(2R)ST1 mutant embryos at stage 11 undergoing cycle 16 exhibit an increased number of mitotic cells, suggesting that cells either enter mitosis prematurely or are delayed in mitosis. Many of these mitotic cells (~30%) show anaphase defects. These included anaphase chromosome bridges and chromosomes apparently severed by the cytokinetic contractile apparatus ('cut' chromosomes) (Quinn, 2001).

Anaphase defects can be caused by a defect in the anaphase promoting complex (APC) mediated proteolysis of chromosome cohesion proteins, which holds sister chromatid centromeres together. Because it was clear that the centromeric regions were separating, whereas the chromosome arms were not in geminin mutant cells, chromosome cohesion defects were considered unlikely. Chromosome segregation defects can also arise because of a defective mitotic spindle. In geminin mutant embryos, however, cells with lagging anaphase chromosomes contain an apparently normal mitotic spindle as visualized using an anti-Tubulin antibody. The anaphase defects may also arise because or a failure to degrade Cyclin B. In geminin mutant embryos, however, the overall pattern of Cyclin B appears normal and in cells exhibiting anaphase defects Cyclin B had already been degraded. Therefore the chromosome segregation defects of the geminin mutant are not attributable to the inability to degrade Cyclin B, to defects in centric chromosome cohesion, or to any visible abnormalities of the mitotic spindle (Quinn, 2001).

Because geminin is maternally supplied and Geminin is very low but not completely absent in cycle 16 geminin mutants, older geminin mutant embryos were examined for over-replication defects. In stage-16 wild-type embryos, endoreplication occurs in the midgut and the dorsal cells. In the geminin mutant, endoreplication occurs in more cells in the midgut and dorsal cell domains and continues in the hindgut and Malpighian tubule domains where it should have ceased. This phenotype is consistent with over-replication occurring in these cells. Furthermore, greater numbers of S-phase cells are observed in the CNS than normal. Many cells appear to label more intensely with BrdU and some are increased in size compared with wild type (Quinn, 2001).

DNA replication was examined in the follicle cells during oogenesis in females trans-heterozygous for the weak, partially female sterile geminin mutant and a strong P allele (l(2)k09107/l(2)k14019). In stage-10B ovaries, the follicle cells of the geminin mutant have switched from general genomic endoreplication to the amplification cycles as normal. In most stage-12 wild-type ovaries, DNA amplification is observed only in the anterior region in one focus per cell, whereas 100% of geminin mutant stage-12 ovaries show strong BrdU labeling of four amplification foci in all follicle cells. By stage 14, all follicle cells of wild-type ovaries have ceased amplification, whereas many follicle cells from 50% of geminin mutant ovaries were still continuing amplification. Therefore, reduced Geminin function in the geminin mutant leads to continued DNA amplification in follicle cells, suggesting that Geminin has an important role in mediating cessation of these replicative cycles (Quinn, 2001).

Xl geminin was identified in a screen for genes that induce neural tissue when overexpressed (Kroll, 1998). To determine whether Drosophila Geminin also induces neural differentiation, geminin was expressed in embryos using En-GAL4 UAS-geminin and these were examined for neural defects by staining with the axonal antibody 22C10. Ectopic overexpression of Geminin in the En stripes results in the formation of ectopic neuronal cells. These ectopic neural cells appear more epidermal than the normal PNS neurons, consistent with the expression of the En driven-geminin in the epidermal cells. Although some disruption in the normal PNS pattern was observed, the ectopic neurons are unlikely to be caused by inappropriate migration of normal neurons because En is not expressed in the PNS and these ectopic neurons were epidermal. Not every Geminin overexpressing cell was induced to form a neuron, perhaps because of cell death, which occurs in some cells when geminin is ectopically overexpressed using the En driver. These data, however, show that overexpression of Geminin is capable of inducing full neural differentiation in at least some cells. Therefore, like Xl Geminin, ectopic overexpression of Geminin is capable of inducing neural differentiation (Quinn, 2001).

Neural differentiation was then examined in l(2)k03202/Df(2R)ST1 mutant embryos by staining with the 22C10 antibody. Although many geminin mutant embryos show a mostly normal 22C10 staining pattern, a small percentage have a striking reduction in 22C10 staining of the dorsal-most peripheral neurons. The variability of this phenotype may be a consequence of maternal Geminin depletion. Further analysis is required to determine whether these neural defects may be a secondary consequence of the cell cycle defects observed earlier in development or to a specific function for Geminin in neural differentiation (Quinn, 2001).

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, RNAi was used to eliminate the Drosophila geminin from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. The silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. The silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, these studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells (Mihaylov, 2002).

Although geminin has recently been implicated in replication licensing in Xenopus egg extract, previous studies have suggested that the depletion of geminin did not cause overreplication in the Xenopus egg extract. The current data for SD2 cells clearly indicate that geminin participates in overreplication control in high eukaryotic cells. One possible explanation for these discrepancies could be that the maternal levels of free Cdt1 in Xenopus egg extract are not significantly affected by geminin depletion. However, it is possible that the control of overreplication in egg extract, which undergoes alternating S phase and mitosis, might be somewhat different from that in cultured SD2 or other somatic cells which show well-defined G1 and G2 phases (Mihaylov, 2002 and references therein).

The phenotype of geminin deficiency is intriguing. The asynchronous and partial overreplication of the genome suggests that the elimination of geminin may result in only a limited capacity for replication of the entire genome and that this replication capacity might be consumed by the replication process itself. Alternatively, geminin may have other functions that limit genome duplication in its absence. For example, geminin may affect Cdt1/Dup localization within the cell or the stability of the Cdt1/Dup protein. Geminin deficiency caused rapid downregulation of its binding partner, Cdt1/Dup. This effect appears to occur at the level of Cdt1/Dup RNA, suggesting that geminin deficiency may cause the downregulation of a factor required for Cdt1/Dup expression. It is possible that Cdt1 transcription is regulated by a checkpoint in response to overreplication. Such a possibility is supported by the observation that the cosilencing of Chk1 had a partial rescue effect on the levels of Cdt1 in geminin-deficient cells. However, these observations do not rule out the possibility that the loss of geminin also affects Cdt1/Dup protein stability or localization in the cell. A recent study suggests that Cdt1 protein, but not RNA, is regulated in a cell cycle-dependent fashion. Cdt1 protein is stable in G1 but is degraded by the ubiquitin-dependent proteolysis upon the entry of S phase. This observation is consistent with the data showing that limited Cdt1 protein is available for each S phase. Geminin knockout may release a limited amount of Cdt1, which is in complex with geminin, promoting the partial overreplication. In addition to the downregulation of Cdt1 RNA, the loss of Cdt1 is partially sensitive to MG132, an inhibitor of 26S proteasome that degrades polyubiquitinated proteins. Thus, in this study, the S phase induced by geminin deficiency may also work to destabilize the Cdt1 protein (Mihaylov, 2002).

In contrast to the effect of geminin deficiency, the silencing of cyclin A caused an initial G2 block followed by duplication of the entire genome, as judged by flow cytometry and morphology studies. This effect is similar to those seen in previous observations of the fission yeast cdc13 mutant, which encodes a mitotic cyclin. However, it is surprising that the silencing of cyclin B did not cause overreplication in SD2 cells. This analysis further suggests that cyclin A deficiency leads to the downregulation of cyclin B, but not vice versa. It has been shown that mutation of the cyclin A gene in Drosophila causes thoracic epidermis cells to skip the mitosis between S phases 16 and 17 and to undergo endoreduplication. The current results are consistent with these observations. In addition, the data unequivocally show that deficiency of cyclin A, unlike that of geminin, causes duplication of the entire genome. Furthermore, these studies indicate that in cyclin A-deficient cells, cyclin B is downregulated. The downregulation of both cyclin A and cyclin B in the cyclin A-deficient cells might explain why overreplication is not observed in the cyclin B-deficient cells, since they still contain relatively normal levels of cyclin A (Mihaylov, 2002).

The data indicate that the silencing of cyclin A induces an overreplication that is quite different from the one caused by geminin deficiency under the assay conditions. The loss of geminin causes only partial overreplication, while the silencing of cyclin A induces the full duplication of the genome. In addition, it appears that geminin deficiency induces substantial cell death while cyclin A silencing does not. The geminin deficiency-induced cell death can be rescued by Cdt1 cosilencing. These observations suggest that mechanisms for suppressing overreplication might be different for geminin and cyclin A. This notion is supported by the finding that the overreplication induced by geminin deficiency may not require the downregulation of cyclin A or cyclin B. Geminin deficiency does not appear to induce a decrease in cyclin A or cyclin B protein levels. Instead, the loss of geminin causes a marked increase in cyclin A and cyclin B protein levels. The overall kinase activity of cyclin B is slightly enhanced with the in vitro histone H1 kinase assay. Conversely, these studies show that cyclin A deficiency does cause a substantial decrease in geminin levels on days 1 and 2 after silencing. However, the downregulation of geminin is still far from complete compared with that caused by geminin knockout. Furthermore, cyclin A deficiency induces the downregulation of Cdt1 at the same time points (days 1 and 2). It thus appears that the ratio of geminin to Cdt1/Dup is not significantly altered by cyclin A deficiency. Moreover, because the silencing of cyclin A causes only G2 arrest on day 1, the downregulation of geminin in cyclin A-deficient cells does not appear to be sufficient to induce overreplication. These studies suggest that other events, independent of or in addition to the downregulation of geminin, may be required for the overreplication induced by cyclin A deficiency (Mihaylov, 2002).

Analyses of cyclin A and geminin double-knockout cells suggest that the loss of cyclin A is dominant over geminin deficiency. In these experiments, even though geminin is completely silenced, coelimination of cyclin A caused only G2 cell cycle arrest on day 1. The cosilencing of cyclin A and geminin on day 2 induced overreplication of the genome which, unlike that induced by geminin deficiency, produced a discrete 8N peak similar to that caused by cyclin A single knockout. These data suggest either that cyclin A is required for subsequent geminin-mediated replication control or that the loss of cyclin A may cause the replication to proceed in a geminin-independent mechanism (Mihaylov, 2002).

Human geminin was originally isolated during the analysis of proteins that are associated with human Chk2 protein. While this interaction appeared to be relatively weak during later verification, attempts have been made to address its potential significance for SD2 cells. In SD2 cells, Chk2 knockout did not have a significant effect on geminin deficiency-induced overreplication or the formation of giant nuclei. However, Chk1/Grapes deficiency significantly suppressed the geminin knockout phenotype, suggesting that Chk1/Grapes possesses a checkpoint function for the overreplication induced by geminin deficiency. This result is consistent with those of previous studies indicating that Chk1/Grapes regulates the DNA replication checkpoint for Drosophila. These studies have shown that interference of Drosophila nuclear division cycles 12 and 13 by X-irradiation or the DNA replication inhibitor aphidicolin activates the Chk1/Grapes signaling pathway. It has been shown that the activated Chk1 kinase phosphorylates Cdc25, promoting its complex formation with 14-3-3 and its subsequent retention in cytoplasm. Consequently, the activated Chk1/Grapes promotes the inhibitory phosphorylation of Cdc2 at threonine14 and tyrosine15 in a Cdc25/String-dependent process. In the current studies, it is likely that overreplication caused by geminin deficiency induces the Chk1/Grapes-mediated checkpoint, leading to the inhibition of the Cdc2 kinase activity and mitosis. This effect may be reflected in part by the observation that cyclin B-associated kinase activity is not dramatically induced by geminin deficiency compared to the marked increase of cyclin B protein levels in these cells. Since the loss of Chk1/Grapes or Cdt1 can either partially or completely rescue the geminin deficiency-induced phenotypes, these studies indicate that the loss of Chk1/Grapes does not suppress geminin deficiency through downregulation of Cdt1. Instead, the loss of Chk1/Grapes partially restores the Cdt1 levels in geminin-deficient cells. In mouse cells, Chk1 deficiency causes an aberrant G2/M cell cycle checkpoint during development or in response to DNA damage, causing the formation of nuclei containing highly condensed and aggregated chromatin and, consequently, massive apoptotic cell death. Although no extensive cell death was observed in Chk1/Grapes knockout SD2 cells, it is possible that Chk1/Grapes silencing allows cells to undergo aberrant mitosis, even though they are overreplicating their genome. This would produce a pseudorescue effect on the geminin deficiency-induced phenotypes. It is unclear how Chk1/Grapes rescues Cdt1/Dup expression. It is possible that Chk1/Grapes may be involved in the suppression of Cdt1/Dup transcription during overreplication. Alternatively, since Cdt1/Dup protein levels are regulated in a cell cycle-dependent fashion, being high in G1 and low in S and G2/M phases, the aberrant mitosis and possibly subsequent G1 phase induced by Chk1/Grapes deficiency may allow Cdt1/Dup to be expressed in G1. Further work is required to clarify these issues. Although no significant effect of Chk2 is seen, Chk2 may play a regulatory role for geminin under certain conditions (Mihaylov, 2002).

Genome instability is often associated with cancer. It is still not clear how these processes are linked to the alteration of DNA replication, mitosis, or G1 cell cycle regulation. The present work suggests that dysregulation of geminin/Cdt1 and cyclin A contributes to genome instability in Drosophila cells. Further studies are necessary to link alterations in the activities of geminin/Cdt1 and mitotic cyclins to human cancer (Mihaylov, 2002).

Metazoans limit origin firing to once per cell cycle by oscillations in cyclin-dependent kinases and the replication licensing inhibitor geminin. Geminin inhibits pre-replication complex assembly by preventing Cdt1 from recruiting the minichromosome maintenance proteins to chromatin. Geminin depletion results in genomic over-replication in Drosophila and human cell lines. Loss of geminin affects other cell cycle-dependent events in addition to DNA replication. Geminin inactivation causes centrosome overduplication without passage through mitosis in human normal and cancer cells. Centrosomes are microtubule-organizing centers that are duplicated during S phase and have an important role in the fidelity of chromosome transmission by nucleating the mitotic spindle. Consistent with this, geminin-depleted cells show multiple mitotic defects, including multipolar spindles, when driven into mitosis by checkpoint abrogation. These results show that the consequences of geminin loss exceed its immediate role in DNA replication and extend to promoting chromosome mis-segregation in mitosis (Tachibana, 2005).

In summary, the results show that loss of geminin has consequences beyond its immediate role in DNA replication. Geminin depletion causes continuous centrosome duplication without passage through mitosis. It is possible that this is a downstream effect of genomic over-replication and activation of the G2-M DNA damage checkpoint pathway, which is implicated in causing aberrations in centrosome number during a prolonged G2 arrest. However, a direct role for geminin in regulating centrosome duplication cannot be excluded at present. Indeed, it is tempting to speculate that geminin functions as a licensing inhibitor of both DNA replication and centrosome duplication during S and G2 phases. Moreover, whereas chemicals that induce genotoxic stress cause centrosome overduplication in the absence of DNA synthesis, geminin depletion leaves the link between DNA replication and centrosome duplication intact. This offers the opportunity to explore further how these crucial cell cycle events are coupled (Tachibana, 2005).


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geminin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 February 2014

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