To test if the cell cycle transcription of double parked (dup) is dependent on E2F, embryos homozygous for a null allele of the dE2F1 subunit, dE2F91 were collected and hybridized with dup riboprobes. The levels of dup transcript are decreased in the endoreplicating gut and appear to be slightly decreased in the CNS. A similar effect on dup transcript is seen in embryos that are homozygous mutant for the other subunit of the E2F transcription factor, of the genotype dDPa2. Thus, dup is a downstream target of the E2F transcription factor. Interestingly, yeast cdt1 transcription is also cell-cycle regulated. Expression of cdt1 is controlled by the G1-S transcription factor Cdc10 that, like E2F, regulates transcription of many genes required for S phase (Hofmann, 1994). This suggests that cell cycle control of dup may be conserved, and Dup may prove to be an important downstream target of E2F in mammalian cells (Whittaker, 2000).
Cyclin E is required to regulate positively the transcription of S phase genes in the nervous system and to downregulate these transcripts in endo cycling cells. The cyclinEl(2)305 and cyclin EPZ5 mutations and a cyclin E deficiency, Df(2L)TE35D1, have similar effects on double parked transcripts. In these embryos, dup is not downregulated properly in the endoreplicating gut such that dup transcripts persist at higher levels than wild type in the anterior midgut, central midgut, and posterior midgut in later embryonic stages. In cyclin E mutant embryos dup transcripts are reduced in the CNS, although not to as great an extent as other S phase genes. Thus, dup expression also is regulated by cyclin E (Whittaker, 2000).
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).
To confirm that the Drosophila Geminin is in fact the Drosophila homolog of vertebrate 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).
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).
Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. MCM10 from Drosophila has been cloned; it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Double parked (Dup or Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. These results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked (Christensen, 2003).
Mcm10 was first identified in S. cerevisiae as defective in S-phase progression and subsequently was shown to be defective in the maintenance of minichromosomes. Work on Mcm10 in S. cerevisiae has revealed that Mcm10 interacts with members of the pre-RC and is required for efficient initiation of DNA replication. Mutants of Mcm10 exhibit pausing of replication forks, suggesting a role for Mcm10 in elongation. Chromatin fractionation experiments show that Mcm10 is constitutively bound to chromatin. Analysis in human has shown that Mcm10 interacts with Orc2, is phosphorylated, and is degraded by an ubiquitin-dependent pathway during the cell cycle. Recent work in Xenopus demonstrates that Mcm10 is required for replication, is dependent on Mcm2-7 for association with the origin, and is necessary for recruitment of Cdc45 (Christensen, 2003 and references therein).
The Drosophila homolog of Mcm10 was first identified by Izumi (2000). The study used the predicted Drosophila Mcm10 to identify homologous human ESTs. The Drosophila Mcm10, known as CG9241, maps to the 2nd chromosome and cytologically to 39B1. Advantage was take of known EST sequences to design primers to amplify MCM10 from cDNA isolated from Drosophila ovary tissue. Sequencing of the resulting clone revealed that Drosophila Mcm10 is a 776 amino acid protein with a predicted molecular mass of 86.5 kDa. Overall, it shares similarity to Human (32.1%), Xenopus (30.5%), Arabidopsis thaliana (29.7%), Caenorhabditis elegans (26.2%), S. cerevisiae (24.1%), and Schizosaccharomyces pombe (23.1%). Alignments of the conserved regions of human, Xenopus, Drosophila, and S. cerevisiae reveal that Drosophila Mcm10 shares a conserved central core and a signature zinc finger motif. In addition, there is a high degree of regional conservation between Mcm10 of Drosophila and higher eukaryotes that points to the fact that studies in Drosophila will have significant bearing on those in Xenopus and human (Christensen, 2003).
Mcm10 has been shown in yeast to interact with all members of the Mcm2-7 family except for the notable exception of Mcm5. In addition, human Mcm10 interacts with Orc2. To investigate which members of the pre-RC interact with Mcm10 in Drosophila coimmunoprecipitation experiments were performed. A stable KC cell line containing Mcm10::GFP was induced or not induced with Cu2+ and cells were harvested, processed, and immunoprecipitated with anti-GFP. Interactions with Mcm10-GFP are detected with Mcm2, Orc2, and the endogenous Mcm10, consistent with the two-hybrid studies. Also probed and shown positive for interactions are Dup, Cdc45, and Hp1. No interaction is detected with Mcm5, and Orc1. Immunoprecipitations were also performed using antibodies to Cdc45, Dup, Mcm2, Orc2, Mcm5, Orc1, and Hp1, respectively, in embryo cell extracts. Similar to the coimmunoprecipitation results with Mcm10-GFP, in all but Mcm5 and Orc1, Mcm10 is detected (Christensen, 2003).
In the absence of a known Drosophila mutant for Mcm10, RNAi was used to determine the function of Mcm10 in Drosophila. RNAi involves addition of dsRNA specific to the mRNA sequence of the target gene. RNAi acts to deplete the mRNA of the target species. The result is that the protein of interest is specifically depleted from cells at a rate corresponding to the inherent stability of the protein. RNAi has been demonstrated as an effective tool in Drosophila tissue culture for determining gene function. In this analysis, KC cells at low densities were inoculated with specific dsRNA and collected over a 5-d period for immunoblot analysis. Over the course of the experiment, cells grew from low to high densities. Low densities corresponded to cell cycle time of ~22 h, and the apparent cell cycle lengthens to 40+ h at high density as cells begin to exit into G0. Mcm10, Cdc45, Mcm2, Mcm5, and Orc2 are all efficiently depleted from KC cells upon addition of specific dsRNA (Christensen, 2003).
Mcm10 and Ccd45 are particularly sensitive to RNAi treatments because both are depleted by 48 h and are undetectable by Western blot. The rapid depletion is indicative of either inherent instability of these proteins or regulation via proteolysis. Mcm2 and Mcm5 show depletion of the bulk of the protein by 48 h but both remain at low levels. In contrast, Orc2 is slowly depleted over the time course compared with the others. This observation is supported by the fact that null mutants for Orc2 in Drosophila persist until third instar, presumably due to the stability of maternal deposits (Christensen, 2003).
Several interesting observations are apparent from these experiments. First, both Mcm10 and Cdc45 exhibit sensitivity to exit into G0 and/or increasing cell densities as shown by the fact that both are reduced in the nonspecific treatment. This is in contrast to Mcm2, Mcm5, and Orc2, which all show increases correlating with increased cell densities and are seen to accumulate as cells exit into G0 and/or increase in density as measured over this time course. These observations suggest that overall stability of Mcm10 and Cdc45 may be regulated as a function of the cell cycle, regulated in relation to cell densities, or a combination of both. In contrast, overall stability of Mcm2, Mcm5, and Orc2 does not seem to be regulated with respect to the cell cycle and/or increased cell densities. The relatively short-lived Drosophila Mcm10 is consistent with observations reported for the human Mcm10. HsMcm10 protein levels are regulated by both phosphorylation- and ubiquitin-dependent proteolysis during late M and early G1 phase. In contrast, S. cerevisiae Mcm10 has been shown to be present at constant levels throughout the cell cycle (Christensen, 2003).
At the outset, one would predict that depletion of proteins required for initiation of DNA replication would have dire consequences for cell growth. Cell growth of KC cells treated with dsRNA specific to Mcm10 and Orc2 was assayed. Over the course of 6 d the growth of cells depleted of Orc2 and Mcm10 seemed unaffected. One could argue that because of the short time assayed and the fact that apparent cell division slows as cell density increases that one would not be able to detect a significant decrease in growth. To address this, a more rigorous test was performed. Cells were diluted every 3 d to keep them at densities that allow logarithmic growth rate. Concurrently, cells were inoculated with specific dsRNA when diluted to ensure that translation of the specific genes did not recover from the initial RNAi treatment. Cell divisions were quantified and cumulative cell divisions calculated over an 18-d period. Cells well past depletion of any detectable Orc2 or Mcm10 divided at wild-type rates. The same phenomenon was observed for depletion of Mcm2, Mcm5, and Cdc45 (Christensen, 2003).
The fact that long-term depletion of Orc2 to <10% had no effect on cell division rates demanded further investigation into how Orc2 depletion was tolerated. Given that KC cells are aneuploidy, it seems reasonable that these cells could tolerate some degree of chromosome loss. Depletion of Orc2, which is involved in initiation of DNA replication, and depletion of Mcm10, which has been shown in yeast to be required for initiation of DNA replication, may have consequences for the DNA content of KC cells (Christensen, 2003).
To investigate possible DNA content effects in Drosophila KC cells depleted of Orc2 and Mcm10 (both positive regulators of DNA replication) were analyzed by FACS. Cells were inoculated with specific dsRNA and maintained in the continual presence of dsRNA and harvested after 10 cell cycles. FACS analysis indicates that both Orc2-depleted and Mcm10-depleted cells show a loss of DNA content compared with controls. This suggests that depletion of Orc2 and Mcm10 both have consequences on the ability of cells to maintain DNA content that may result from decreases in the efficiency of DNA replication or chromosome stability (Christensen, 2003).
RNAi is specific to the target protein. However, because depletion of a particular protein does not occur in a vacuum but rather in a network of interactions, Mcm10 stability was examined in cells depleted of other proteins. KC cells were diluted in the presence of specific dsRNA for ~10 cell cycles. Cells were harvested, lysed, and whole cell extracts were loaded onto SDS-PAGE gels. Blots were then probed for Mcm10, Cdc45, Mcm2, Mcm5, Orc2, Dock, and lamin. Total Mcm10 protein levels are reduced in KC cells depleted of Mcm2, Orc2, and to a lesser extent Mcm5. Cdc45 levels are reduced when Mcm10, Mcm2, and Mcm5 are depleted. Mcm2 levels are slightly reduced when Orc2 is depleted. Last, Orc2 levels are relatively unchanged in all but the specific treatment (Christensen, 2003).
Depletion of Mcm10 or Cdc45 results in strikingly similar defects in chromosome morphology. The condensation defects apparent in both depletions consist of similar 'dumbbell' shaped chromosomes. Sister chromatid separation is observed in both treatments with a higher frequency observed in cells depleted of Cdc45. Chromosome fragmentation in Mcm10 and Cdc45 depletions is observed at levels no higher than that of wild type and is likely a consequence of specimen preparation. The fact that these defects are so similar combined with the findings that these two proteins have been shown to interact supports the supposition that these two proteins function in concert in the same pathway (Christensen, 2003).
Depletion of Mcm2, Mcm5, or Orc2 results in a high degree of fragmentation in addition to lateral condensation defects suggestive of incomplete DNA synthesis and subsequent unchecked chromosome separation. Mcm2 depletion demonstrates the most severe defect with no wild-type figures found and an overwhelming percentage in class III, the most severe class. Sister chromatid separation is noted in all three treatments but is more prevalent in Mcm2-depleted cells. The small discrepancies between Mcm2 and Mcm5 depletions with respect to severity may represent merely different stability levels of the protein since Mcm2 is depleted more rapidly and thoroughly by RNAi (Christensen, 2003).
Thus Drosophila Mcm10 biochemically interacts with components of the pre-RC, including Mcm2, Orc2, and Dup, and with itself. These interactions further the argument that Mcm10 is present in the pre-RC before the transition to the pre-IC and that interaction of Mcm10 with its various partners may be mediated by a Mcm10 multimer Christensen, 2003).
This study presents the first evidence for an interaction between Mcm10 and Double parked (Dup), the Drosophila homolog of Cdt1. This interaction is of particular interest in light of the observation that Cdt1 is required for loading of the MCM complex and is a target for geminin regulation. These and other studies suggest that Cdt1 interacts with the ORC and MCM complexes. In this context, Mcm10 may be interacting directly with Dup or indirectly through ORC or the MCMs to facilitate steps in pre-RC assembly (Christensen, 2003 and references therein).
Mcm10 interaction with Cdc45 has been implied previously in Xenopus where it has been shown that Cdc45 requires Mcm10 for origin binding. In addition, it has been shown that S. cerevisiae Mcm10 genetically interacts with Cdc45. The experiments presented in this study extend the interaction between Cdc45 and Mcm10 to Drosophila and support the hypothesis that stabilization of Cdc45 binding to origins may occur via a direct or indirect interaction with Mcm10 (Christensen, 2003).
Heterochromatin protein 1 (Hp1) has been shown to interact with members of the ORC complex. Interaction with Mcm10 suggests that, like Orc2, Mcm10 may be associated with heterochromatin to facilitate the role of Hp1 in heterochromatin formation, maintenance, transcriptional repression, or epigenetic inheritance. An interaction between Mcm10 and Hp1 may point to a trend that more members of the pre-RC are involved in heterochromatin formation. Indeed, this process may be a fundamental function of prereplication proteins. The involvement of ORC in establishing silencing at the mating type loci in yeast has been pointed to as an analog to the function of ORC in establishment of heterochromatin in Drosophila. In fact, the function of ORC in silencing and heterochromatin formation may be the most conserved aspect of ORC function. Drosophila Orc2, for example, is able to complement the silencing defect of a mutant Orc2 allele in yeast, but it is unable to complement the replication defect. In addition to ORC, Cdc45 and Mcm2 have been implicated in yeast to be involved in chromatin formation. The implication that Mcm10 is involved in formation of heterochromatin in Drosophila by virtue of an interaction with both Orc2 and Hp1 raises the tantalizing possibility that these dynamic properties of chromatin may require not only ORC function but also a number of other prereplication proteins as well (Christensen, 2003).
Key members of the pre-RC and pre-IC are effectively and specifically depleted from KC cells by RNAi. Although deficiencies in DNA replication were not directly tested, replication must still be occurring at a level sufficient to maintain growth rates under long-term depletion of Mcm10 and Orc2. Precedence for this phenomenon has been reported in human HCT116 colon carcinoma cells where 10% of the wild-type level of Orc2 are sufficient to sustain normal chromosomal replication. Maintenance of DNA content at levels that permit cell viability may be due to these proteins being required in very small amounts combined with the hypothesis that few origins are required to replicate the genome. The results suggest that RNAi generates severe hypomorphs for protein depletions and not total depletion (Christensen, 2003).
No explanation is available for the mechanism that selectively retains a complement of chromosomes that ensures viability as a result of reduced DNA replication. Precedence for ploidy effects by depletion of proteins by RNAi in Drosophila SD2 cells has recently been reported for both geminin and Dup. This study reports that depletion of geminin, a negative regulator of DNA replication, resulted in an increase in DNA content. In contrast, depletion of Dup, a positive regulator of DNA replication, results in loss of DNA content. Defects in the growth of these cells were not reported (Christensen, 2003).
Protein levels of certain members of the pre-RC may be coupled. These observations could be due to at least three different factors or combinations of factors. (1) Depletion of one protein results in transcriptional repression of another either directly or indirectly. (2) Some or all of these depletions result in cell cycle defects that have consequences for other proteins, although cell cycle length is unchanged. (3) Interactions between proteins are required for stability. In other words, proteins are stable in a complex but not individually. An example of this is the coupling of Cdt1 and geminin protein levels observed when geminin is depleted from tissue culture. Depleting cyclin A also results in a corresponding decrease in cyclin B protein levels. Another case of association-dependent stabilization is the destruction of Cdc6 when removed from chromatin and association with the pre-RC (Christensen, 2003).
This is the first report of a possible role for Mcm10, Mcm2, Mcm5, and Cdc45, respectively, in proper condensation of chromosomes. This study demonstrates the utility of RNAi in tissue culture cells for assaying chromosomal condensation defects. Indeed, it points the way to analysis of other known replication proteins for which mutants do not yet exist with respect to functions in condensation. An interesting point to consider is that the process of chromosome condensation may be more sensitive to the dosage of these proteins than is DNA replication, because cells remain viable over long-term depletion. These observations raise the intriguing possibility that the bulk of these proteins in cells may function in chromosome condensation pathways and not overtly participate in the initiation of DNA replication. Alternatively, the depletion of these proteins may simply reduce the number of initiation sites along the chromosome, resulting in fewer replication foci. This reduction in foci may have direct mechanistic consequences for condensation. A third possibility is that depletion of replication initiation factors in S phase may force cells to enter mitosis prematurely, resulting in aberrant chromosome condensation. Indeed, depletion of Cdt1/Dup protein first shown in S. pombe and more recently shown in Drosophila results in chromosome condensation without DNA replication and thereby bypassing S phase. It is not believed that the third possibility is a likely explanation because no dramatic loss of cell viability or a change in cell cycle length was observed as expected of mitosis without S phase. Determining whether these proteins are directly linked to condensation or are merely linked via DNA replication is a question that remains to be answered (Christensen, 2003).
It is becoming increasingly clear that proteins involved in DNA replication are necessary for establishment of proper chromosomal condensation. There is some debate as to whether the defects observed are due to the specific functions of individual proteins or are a general function of compromised DNA replication. The addition of Mcm10, Mcm2, Mcm5, and Cdc45 to the repertoire of replication proteins required for proper chromosomal condensation lends support to the hypothesis that DNA replication and condensation are generally linked (Christensen, 2003).
What is the mechanism by which replication is linked to condensation? At the outset, it seem reasonable that organization of chromatin would happen in concert with replication. Spatially and temporally separating the processes would seem to be both inefficient and problematic with respect to entanglement of DNA and nuclear organization. A simple mechanism for linkage of replication to condensation has been put forth that suggests that the density of replication initiation along a chromosome and the resulting DNA replication foci has impact, on a primary level, on the lateral condensation of a metaphase chromosome. This hypothesis fits very well with the 'dumbbell' lateral condensation defects observed when replication proteins are depleted (Christensen, 2003 and references therein).
The simple mechanistic model probably has relevance to the linkage of condensation to DNA replication but may not provide a complete picture as to the role of pre-RC proteins in this process. There are several observations that speak to the possibility that the proteins of the pre-RC have roles outside of DNA replication. A comparison of two recent studies in yeast looking at global binding of prereplication complexes and global origin usage reveals that there are 30% less active origins compared with those predicted by binding of pre-RC proteins. These observations point to the fact that sites not used for initiation are occupied by members of the pre-RC, leaving open the possibility for some functional role for these assemblies in chromatin condensation (Christensen, 2003).
This study has presented evidence for the conservation of Mcm10 function from Drosophila to S. cerevisiae. Drosophila Mcm10 interacts with known members of the pre-RC, consistent with a role in the assembly of the pre-RC. Moreover, Mcm10 interacts with Cdc45, suggesting that Drosophila Mcm10 may also participate in the transition to the pre-IC. Further evidence for a role of Mcm10 in DNA replication comes out of the observation that depletion of Mcm10 by RNAi, similar to that of Orc2, resulted in a loss of DNA content. Mcm10 is also required for proper chromosome condensation. This role may be facilitated by an interaction with Hp1 (Christensen, 2003).
It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. The developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms, has been studied. Phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, these results indicate that stringent regulation of Dup protein is critical to protect genome integrity (Thomer, 2004).
To determine whether oscillation of Dup protein levels during cell cycles is due to Dup protein degradation at G1/S, Dup expression within the synchronized cell cycles of the larval eye primordium was examined. Late in third instar, a wave of differentiation sweeps across the eye imaginal disc, which is visible as a morphogenetic furrow (MF). Cells are synchronized in G1 upon entering the furrow. Specific cells posterior to the furrow then enter a synchronous S phase, which is visible as a stripe of BrdU labeling. Labeling with affinity-purified rabbit polyclonal Dup antibody indicates that the protein is abundant in nuclei of late G1 cells, but is undetectable in S phase cells incorporating BrdU. Labeling with a guinea pig anti-Dup antibody gave identical results suggesting that immunolabeling reflects Dup protein in vivo. Double labeling for Dup and cyclin E indicates that both are abundant in nuclei of cells in late G1, but then Dup rapidly declines while cyclin E persists into S phase. Labeling for the G2 and M phase marker cyclin B also indicates that Dup levels decline significantly before cells enter G2. Similar results were obtained for the non-synchronized cell cycles in the eye and other imaginal discs. This rapid decline in protein is primarily due to post-transcriptional regulation because in situ hybridization indicates that dup mRNA persists after G1. Moreover, expression of a dup transgene from the strong hsp70 promoter does not result in detectable Dup protein during S phase. The data suggest that, similar to Cdt1 in humans and other organisms, Dup protein is abundant in G1 when origins are licensed, but is then rapidly degraded when cyclin E appears at G1/S (Thomer, 2004).
Beginning in late mitosis, origins of replication are prepared for replication by binding of a pre-replicative complex (pre-RC), which is subsequently activated to initiate replication at the onset of S phase. The building of the pre-RC onto origins in late mitosis/early G1 is a stepwise process. The origin recognition complex (ORC) serves as a scaffold for subsequent association of Cdc6 and Cdt1, both of which are required to load the Minichromosome Maintenance (MCM) complex, the replicative helicase. Once MCMs are loaded, the origin is considered to be licensed for subsequent replication. Cdc7 kinase, with its activating subunit Dbf4, and CDK2 kinase, activated by cyclin E or cyclin A, are then required for initiation of replication. Initiation is associated with departure of Cdc6, Cdt1, MCMs, and, in multicellular eukaryotes, certain ORC subunits from the origin. Continued CDK activity in S, G2, and early M phases inhibits reassembly of the pre-RC to block origin refiring. Unique to multicellular eukaryotes is another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex. It is only after Geminin and cyclins are degraded at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes (Thomer, 2004 and references therein).
Although phosphorylation of pre-RC subunits appears to be important for initiation and to block pre-RC re-assembly, the biochemical mechanisms are not fully understood. In the yeasts Saccharomyces cerevisiae and S. pombe, CDKs block re-replication by phosphorylating several pre-RC targets including CDC6 and subunits of the ORC and MCM complex. All three of these blocks must be abrogated before even partial re-replication is permitted in S. cerevisiae cells in G2, suggesting that multiple reinforcing mechanisms have evolved to protect the integrity of the genome. In S. pombe, however, over-expression of Cdc18 (the Cdc6 homolog) alone, but not other pre-RC subunits, is sufficient to induce re-replication. Thus, whether mis-regulation of a single protein can induce re-replication may differ among organisms. In higher eukaryotes, it also appears that CDKs block re-replication by targeting multiple pre-RC subunits to protect genome integrity (Thomer, 2004 and references therein).
Despite the prevailing concept of redundant controls, recent evidence suggests that regulation of Cdt1 is especially important to inhibit re-replication. In a number of systems, over-expression of Cdt1, or inactivation of its inhibitor Geminin, causes partial, but not full, re-replication of the genome. In all organisms examined, except S. cerevisiae, the majority of Cdt1 protein is rapidly degraded at the G1/S transition. Evidence from several organisms suggests that Cdt1 is targeted for degradation at the proteasome by two ubiquitin ligase complexes, an SCF (Skp1, Cul1, F box) ubiquitin ligase that contains the specificity subunit Skp2, and an SCF-like ubiquitin ligase that is based on Cul4 (Higa, 2003; Li, 2003; Nishitani, 2001; see Drosophila Cul4). This degradation is probably important because over-expression of Cdt1 in p53 mutant human cells in culture can lead to partial re-replication, and contributes to oncogenic transformation of mouse erythroid cells (Arentson., 2002; Vaziri, 2003). In Caenorhabditis elegans, RNAi of Cul4 leads to stabilization of Cdt1 protein and polyploidization (Zhong, 2003). It is unclear, however, whether Cul4 controls degradation of other proteins important for re-replication control. Therefore, two important remaining questions are whether increased Cdt1 protein is sufficient to induce genome reduplication in normal cells during development, and what coordinates the rapid degradation of Cdt1 with the initiation of DNA replication at the G1/S transition (Thomer, 2004 and references therein).
The Drosophila ortholog of Cdt1, the double-parked (dup) gene, was initially identified as recessive embryonic lethal or female-sterile mutants that have defects in genomic replication or developmental amplification of eggshell (chorion) protein genes in the ovary. Evidence is provided that degradation of Dup is controlled in part by cyclin E/CDK2 phosphorylation, and additional mechanisms also ensure Dup degradation. Control of Dup protein abundance is critical because increased expression of Dup in diploid cells is sufficient to induce polyploidization and cell death in developing tissues. Interestingly, over-expression of wild-type and mutant Dup derivatives have different effects on genomic replication than on amplification from chorion origins. These last results provide insight into how phosphorylation regulates Dup during these developmentally distinct replication programs, and suggest that Dup participates in replication fork elongation during amplification (Thomer, 2004).
Therefore, phosphorylation and stability of Dup depends on cyclin E/CDK2 activity. It is likely that part of this regulation is direct because Dup associates with CDK2 protein and activity in embryos. The results of the mutagenesis show that the N terminus of Dup is necessary and sufficient for degradation at G1/S. Mutation of the CDK sites in the N terminus, however, only partially stabilize the protein, suggesting the existence of other CDK2-dependent mechanisms for degradation. It is crucial to tightly regulate the abundance of Dup protein because its over-expression is sufficient to induce a full genome reduplication and cell death in the ovary and imaginal discs. The different effects on amplification and genomic replication suggest that phosphorylation of the N terminus of Dup protein may be required for replication fork elongation during amplification and provides insight into the mechanism of this developmentally specific replication program (Thomer, 2004).
The results suggest that cyclin E/CDK2 phosphorylates the Dup N terminus contributing to its instability at G1/S. Dup was degraded during periodic endocycle S phases that are solely regulated by oscillating cyclin E/CDK2, further supporting a link between this kinase and Dup degradation. Although the N terminus was necessary and sufficient for degradation, mutation of the ten N-terminal CDK sites within Dup 10(A) only partially stabilized the protein. This suggests that there are other cyclin E/CDK2-dependent mechanisms that trigger Dup degradation independent of these ten sites during S phase. It has been noted that the C terminus of Dup contains a PEST sequence, and there are several serines and threonines in the C terminus that are potential targets of phosphorylation. Although the requirement for these sites has not been directly tested, the stability of C-Dup indicates that they are not sufficient for degradation at G1/S. To explain these results, a bi-phasic degradation model is suggested where cyclin E/CDK2 phosphorylation promotes Dup degradation in late G1, whereas other fail-safe mechanisms become operative only during S phase. This would explain why inhibiting CDK2 and S phase entry with GMRp21 completely blocked Dup degradation (Thomer, 2004).
A number of recent publications describe results for Cdt1 in human cells that are similar to those in flies. These results suggest that cyclin A/CDK2 phophorylates the human Cdt1 N terminus, which enhances its binding to the Skp2 subunit of the SCF ubiquitin ligase. Like Dup, non-phosphorylatable Cdt1 mutants are only partially stabilized, but simultaneously inhibiting CDK2 and S phase entry with p21 completely blocks degradation. Previous evidence in C. elegans, human and Drosophila cells have suggested that destruction of Cdt1 may be mediated by two ubiquitin ligases, an SCF complex containing Skp2, and an SCF-like complex based on Cul4. For many substrates of the SCF, prior phosphorylation is required for their subsequent recognition and ubiquitinylation, including substrates phosphorylated by CDK2 at G1/S. It is not known whether prior phosphorylation is required for substrate recognition by Cul4-based ubiquitin ligases. It is tempting to speculate, therefore, that the bi-phasic degradation of Cdt1 that may reflect its modification by two distinct ubiquitin ligases: a phosphorylation-dependent ubiquitinylation by the SCF complex, and a phosphorylation-independent ubiquitinylation by a Cul4-based complex. Clearly, more experiments are needed to sort out the complexity of this regulation. Nonetheless, the similar results from flies and humans suggest that tight regulation of Cdt1 abundance is a generally conserved and important mechanism to protect genome integrity in eukaryotes (Thomer, 2004).
CDK activity and Geminin play central roles in the block to re-replication. The results reported in this study show that Dup over-expression is sufficient to induce a full genome reduplication in normal cells in developing tissues, transforming diploid into polyploid cells. This phenotype is more profound than that of Geminin mutants, suggesting that degradation of Dup protein is of highest priority to protect genome integrity. An important caveat is that in these experiments Dup is over-expressed and therefore not equivalent to an absence of degradation. It was found, however, that even small, undetectable increases in Dup protein can have profound consequences. Moreover, after multiple heat pulses, Dup protein was undetectable during S phase, yet it induced extensive re-replication in most cells. The prolonged genomic replication in the synchronized cells of the eye disc suggests that this small increase in Dup protein may permit origins to be relicensed and reinitiate within a single S phase. While the precise molecular mechanism for how increased Dup promotes re-replication remains undefined, the results indicate that even a small increase in Dup protein is sufficient to compromise genome integrity (Thomer, 2004).
The other phenotype associated with over-expression of Dup is cell death. Dup 10(A), created by mutating the serines and threonines at putative phosphorylation sites to alanine, causes more cell death than wild-type Dup, suggesting that phosphorylation of the Dup N terminus influences this phenotype. In human cells re-replication due to over-expression of Cdt1 is more easily detected when p53 is mutant, probably because they escape apoptosis triggered by re-replication. Therefore the model is favored that Dup over-expression induces re-replication, which in turn can lead to the activation of checkpoints and apoptosis (Thomer, 2004).
In recent years, the analysis of replication from the defined chorion amplification origins has been a prominent genetic and molecular model system for the regulation of DNA replication in metazoa. Chorion origins require pre-RC proteins, cyclin E/CDK2 and Dbf4/Cdc7 kinases, indicating that their regulation resembles that of genomic origins. They clearly differ, however, in that they re-replicate at a time when no other origins are firing, and understanding this exception should provide insight into the rules of regulation of all origins. Surprisely, the carboxyl-terminal half of Dup, although having no effect on genomic replication, is a hyperactive protein that causes over-amplification from chorion origins. The Dup 10(A) mutant gave the opposite result; it was dominant negative and strongly inhibited amplification. It is proposed that during amplification phosphorylation of the Dup N terminus abrogates its inhibition of the activity of the C terminus, explaining why deleting the N terminus results in a hyperactive protein, whereas blocking its phosphorylation results in an inactive protein. An important functional role for the C terminus is consistent with its binding to MCM proteins, and the fact that among Cdt1 family members the C-terminal half is much more highly conserved than the N-terminal half of the protein. Most Cdt1 proteins have known or potential CDK phosphorylation sites in their N terminus, despite its poor conservation, supporting the notion that its conserved function is to mediate regulation by CDKs (Thomer, 2004).
The different effects on amplification versus genomic replication suggest a distinction in the regulation or function of Dup in these two processes. It has been proposed that Dup participates in fork elongation during amplification, based on immunolabeling at chorion foci. This study shows that in other cell cycles Dup is rapidly degraded at the onset of S phase and not present during fork elongation, similar to results from human and other cells. Moreover, S. cerevisiae cells experimentally depleted of Cdt1 within S phase are able to complete genomic replication, inconsistent with a role in elongation. Expression of Dup 10(A), however, inhibits BrdU incorporation within 1 hour in all stages of amplification, including late stages when only elongation of forks is occurring. This rapid and complete inhibition of BrdU incorporation by Dup 10(A) cannot be an indirect effect of origin inhibition, and supports the proposed role for Dup at the replication fork. Furthermore, this suggests that phosphorylation is important for the function of Dup in elongation during amplification. The distinct activities of C-Dup and Dup 10(A) in genomic replication versus amplification provide a molecular handle on the mechanism by which these two developmental replication programs differ, possibly resulting from the activity of Dup at the fork. The function of Dup at the fork may be related to its known ability to load the MCM complex helicase onto chromatin. It also raises the possibility that Cdt1 family members may act at the fork under other special circumstances (Thomer, 2004).
The CUL4 (see Drosophila Cul4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate cell cycle, DNA replication and DNA damage response. To determine the composition of CUL4 ubiquitin E3 ligase complex, anti-CUL4 antibody affinity chromatography was used to isolate the proteins that associated with human CUL4 complexes, and they were identified by mass-spectrometry. A novel and conserved WD40 domain-containing protein, the human homologue of Drosophila Lethal(2) denticleless protein (L2DTL) or fission yeast CDT2, was found to associate with CUL4 and DDB1. L2DTL also interacts with replication licensing protein CDT1 in vivo. Loss of L2DTL in Drosophila S2 and human cells suppresses proteolysis of CDT1 in response to DNA damage. The human L2DTL complexes weere isolated by anti-L2DTL immuno-affinity chromatography from HeLa cells, and it was found to associate with DDB1, components of the COP9-signalosome complex (CSN) and PCNA. PCNA interacts with CDT1 and loss of PCNA suppresses CDT1 proteolysis after DNA damage. These data also revealed that in vivo, inactivation of L2DTL causes the dissociation of DDB1 from the CUL4 complex. These studies suggest that L2DTL and PCNA interact with CUL4/DDB1 complexes and are involved in CDT1 degradation after DNA damage (Higa, 2006).
CUL4/ROC1 E3 ligase targets CDT1 for ubiquitin-dependent proteolysis in response to UV or gamma-irradiation (Higa, 2003). Since the CUL4 complex represents a new type of cullin-containing E3 ligases that is very different from the SCF, CUL2 and CUL3 ubiquitin E3 ligase complexes, attempts were made to determine the protein composition of the CUL4 complex to understand the mechanism of substrate recognition and its regulation. To identify these proteins, anti-CUL4 antibody affinity chromatography was used to isolate CUL4 complexes from human cells. Mass-spectrometry analysis revealed that the isolated human CUL4B complex from HeLa cells contained DDB1, components of the CSN complex, and the human homologue of Drosophila lethal(2) denticleless protein (Kurzik-Dumke, 1996). L2DTL is a conserved WD40 repeat-containing protein which is essential for Drosophila embryogenesis but without any known molecular function. L2DTL also shares homology with the S. pombe CDT2. To further determine the interaction, rabbit polyclonal antibodies were raised against human L2DTL protein (Higa, 2006).
Coimmunoprecipitation performed with L2DTL antibodies confirmed that human L2DTL indeed interacts with endogenous CUL4A, CUL4B and DDB1 in human cells. While the anti-L2DTL antibodies consistently coimmunoprecipitated DDB1, the interaction between L2DTL and CUL4A or CUL4B varies between experiments, suggesting L2DTL may preferentially interact with DDB1. However, the lack of a DDB1 antibody that can immunoprecipitate the CUL4/DDB1/L2DTL complexes prevented further exploration of this possibility. Antibody was also raised against the Drosophila L2DTL protein. The data indicate that the Drosophila L2DTL protein can be detected in the anti-CUL4 immuno-complex in Drosophila Schneider D2 (S2) cells (Higa, 2006).
Structurally, the L2DTL protein contains a conserved WD40 repeat region at its amino terminus and a less conserved carboxy half. To determine the region in L2DTL that mediates its interaction with CUL4 and DDB1, deletion mutants were made that lack either the WD40 repeats or the carboxy terminal region, and their binding to CUL4 or DDB1 was analyzed. While wild-type L2DTL binds both CUL4 and DDB1, loss of either the WD40 repeats or the carboxy terminal region severely impairs the association between L2DTL and CUL4 or DDB1, suggesting that both domains are required for L2DTL interaction with DDB1-CUL4 complexes (Higa, 2006).
One of the best characterized CUL4 substrates is replication licensing protein CDT1, To determine L2DTL function, the expression of Drosophila L2DTL was silenced in S2 cells by RNA interference (RNAi) because of highly efficient gene silencing in these cells. The effect of L2DTL inactivation on CDT1 proteolysis induced by gamma-irradiation was examined. While CDT1 is rapidly degraded in response to gamma-irradiation in S2 cells treated with control dsRNA, inactivation of L2DTL by RNAi completely blocked CDT1 proteolysis after radiation. This effect is similar to inactivation of CUL4 on CDT1 in parallel experiments, indicating that Drosophila L2DTL is required for CDT1 proteolysis in response to DNA damage (Higa, 2006).
Replication licensing protein CDT1 serves as a substrate of CUL4 ubiquitin E3 ligase complex. The inactivation of Drosophila L2DTL suppresses CDT1 proteolysis in response to DNA damage. Therefore whether L2DTL can interact with CDT1 was tested. Since Drosophila anti-L2DTL antibodies do not immunoprecipitate L2DTL complex, the interaction between L2DTL and CDT1 was characterized in human cells. L2DTL can be detected in the anti-CDT1 immunocomplexes while CDT1 is also present in anti-L2DTL complexes, albeit the signal is relatively weak under the immunoprecipitation conditions used. These data indicate that there is an interaction between the endogenous L2DTL and CDT1 proteins. These studies are consistent with observations CUL4 and its associated DDB1 interact with CDT1 in vivo (Higa, 2006).
Because of the relative weak interaction between L2DTL and CDT1 under various conditions, the possibility was considered that there may be additional subunits in the CUL4 complexes that regulates CDT1 stability. To further identify the proteins that associate with L2DTL and CUL4 complexes, L2DTL complexes were affinity purified using anti-L2DTL antibodies as the affinity resins for chromatography from HeLa cell lysates. Mass-spectrometry was used to analyze proteins that are specifically associated with L2DTL complexes. Peptides corresponding to DDB1 and subunits of the CSN complex were obtained from the isolated protein bands in L2DTL complexes. One of the peptides in L2DTL complexes corresponds to PCNA. To confirm these interactions, immunoprecipitation of cell lysates prepared from various human cells was performend, followed by western blotting using anti-L2DTL, CSN5 and PCNA antibodies. The data confirmed that endogenous CSN5 and PCNA proteins are indeed present in L2DTL complexes isolated from various mammalian cells (Higa, 2006).
To determine whether L2DTL functions to regulate CDT1 stability in human cells, the expression of human L2DTL was silenced in HeLa, U2OS, or other human cell lines by small interfering RNA (siRNA). Loss of human L2DTL in these cells also suppresses CDT1 degradation in response to gamma-irradiation. These studies demonstrate that L2DTL is a novel protein that associates with CUL4 and DDB1 and is required for CDT1 degradation in response to DNA damage in both Drosophila and human cells. Sometimes, it was observed that CDT1 is stabilized in L2DTL silenced cells in the abscence of irradiation. This effect was also sometimes observed in DDB1 or CUL4 silenced cells. Since the protein stability of CDT1 is regulated in S phase it is possible that L2DTL and DDB1/CUL4 complexes regulate CDT1 proteolysis in S phase cells (Higa, 2006).
Since inactivation of L2DTL prevents CDT1 degradation in response to DNA damage, the expression of PCNA was also silenced in human cells. Similar to L2DTL or DDB1 silenced cells, inactivation of PCNA by siRNA prevented CDT1 proteolysis in response to gamma irradiation. Whether CDT1 interacts with PCNA was also tested. The recombinant CDT1 and PCNA directly interact in insect SF9 cells infected with baculoviruses encoding CDT1 and PCNA cDNAs. These studies suggest that PCNA is involved in regulating DNA-damage induced proteolysis of CDT1. These data suggest that CDT1 proteolysis after DNA damage requires the presence of L2DTL, PCNA and the DDB1/CUL4 E3 ligase complexes (Higa, 2006).
Since L2DTL binds to DDB1 and CUL4, the mechanism for the requirement of L2DTL by the CUL4 complex was explored. It was found that loss of L2DTL by siRNA sometimes reduces the binding of DDB1 to CUL4 complexes. These observations suggest that one function of L2DTL may be to facilitate the interaction between DDB1 and CUL4A complexes in vivo (Higa, 2006).
Therefore a novel WD40 repeat-containing protein, L2DTL, binds to DDB1, CUL4, PCNA and CSN. These studies further indicate that PCNA associates with L2DTL and CDT1. Similar to the effect of loss of CUL4 and DDB1, inactivation of either L2DTL or PCNA prevented CDT1 proteolysis in response to DNA damage. In contrast, inactivation of CSN5 or CSN2, components of CSN complex, by siRNAs did not alter CDT1 proteolysis after DNA damage, even though the protein levels of CSN2 or CSN5 were substantially reduced. This differs from previous data, which demonstrated loss of CSN complex abolished CDT1 degradation in Drosophila S2 cells after gamma-irradiation. It is possible that the siRNAs used against human CSN2 or CSN5 were still not sufficient to silence the expression and the activity of CSN to the level that can impact CDT1 proteolysis. Alternatively, because cullin deneddylation can be mediated by CSN and DEN1 it is possible that the function of CSN and DEN1 may overlap. Additional studies also showed that CUL4A, DDB1, L2DTL and PCNA also interact with p53 and MDM2 in human cells and are required for the CUL4-mediated p53 polyubiquitination activity. These studies suggest that L2DTL and PCNA may be part of the CUL4 complexes that regulate the protein stability of CUL4 substrates such as CDT1 and p53. In this regard, it was found that loss of L2DTL often leads to the dissociation of DDB1 from CUL4 complex. It is possible that L2DTL may play a role in promoting and/or stabilizing the interaction between DDB1 and CUL4 complex. Consistently, the fission yeast CDT2 was recently isolated as a CUL4 binding protein. Since L2DTL and PCNA interact with CDT1 and p53/MDM2, these studies suggest that L2DTL and PCNA also contribute to substrate recognition of the DDB1/CUL4 E3 ligase complex (Higa, 2006).
The proper execution of premeiotic S phase is essential to both the maintenance of genomic integrity and accurate chromosome segregation during the meiotic divisions. However, the regulation of premeiotic S phase remains poorly defined in metazoa. Here, this study identified the p21Cip1/p27Kip1/p57Kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) as a key regulator of premeiotic S phase and genomic stability during Drosophila oogenesis. In dap-/- females, ovarian cysts enter the meiotic cycle with high levels of Cyclin E/cyclin-dependent kinase (Cdk)2 activity and accumulate DNA damage during the premeiotic S phase. High Cyclin E/Cdk2 activity inhibits the accumulation of the replication-licensing factor Doubleparked/Cdt1 (Dup/Cdt1). Accordingly, this study found that dap-/- ovarian cysts have low levels of Dup/Cdt1. Moreover, mutations in dup/cdt1 dominantly enhance the dap-/- DNA damage phenotype. Importantly, the DNA damage observed in dap-/- ovarian cysts is independent of the DNA double-strands breaks that initiate meiotic recombination. Together, these data suggest that the CKI Dap promotes the licensing of DNA replication origins for the premeiotic S phase by restricting Cdk activity in the early meiotic cycle. dap-/- ovarian cysts frequently undergo an extramitotic division before meiotic entry, indicating that Dap influences the timing of the mitotic/meiotic transition (Narbonne-Reveau, 2009).
During the meiotic cycle, germ cells complete two divisions to produce haploid gametes. Before the two meiotic divisions, the germ cells duplicate their genomes during the premeiotic S phase. Events unique to the premeiotic S phase, such as the expression of REC8, a member of the kleisin family of structural maintenance of chromosome proteins, are required for the full execution of the downstream meiotic program. How this specialized meiotic S phase is regulated, as well as how similar it is to the mitotic S phase, has long been a question of interest. Studies from yeast indicate that the mitotic cycle and the meiotic cycle use much of the same basic machinery to replicate their genomes. For example, the minichromosome maintenance complex (MCM2-7), which functions as a DNA replication helicase, is essential for the duplication of the genome during both the mitotic and premeiotic S phase. Additionally, both the mitotic and premeiotic S phase require the activity of cyclin-dependent kinases (Cdks). Yet, despite its fundamental importance to both the maintenance of genomic integrity and the downstream events of meiosis, little is known about the regulation of premeiotic S phase metazoa (Narbonne-Reveau, 2009).
Drosophila provides an excellent model to examine the early events of the meiotic cycle, because the entire process of oogenesis takes place continuously within the adult female. In Drosophila, each ovary is composed of 12-16 ovarioles containing linear strings of maturing follicles also called egg chambers. New egg chambers are generated at the anterior of the ovariole in a region called the germarium that contains both germline and somatic stem cells. The germarium is divided into four regions according to the developmental stage of the cyst. Oogenesis starts in region 1 when a cystoblast, the asymmetric daughter of the germline stem cell, undergoes precisely four round of mitosis with incomplete cytokinesis to produce a cyst of 16 interconnected germline cells with an invariant pattern of interconnections (individual cells in the cyst are referred to as cystocytes). Stable actin-rich intercellular bridges called ring canals connect individual cystocytes within the cyst. Germline cyst formation is accompanied by the growth of the fusome, a vesicular and membrane skeletal protein-rich organelle that forms a branched structure extending throughout all the cells of the cyst. After the completion of the mitotic cyst divisions, all 16 cystocytes complete a long premeiotic S phase in region 2a of the germarium. Subsequently, the two cystocytes with four ring canals form long synaptonemal complexes (SCs) and begin to condense their chromatin, suggesting that they are in pachytene of meiotic prophase I. Several of the cells with three ring canals also assemble short SCs, and even cells with only one or two ring canals are occasionally seen to contain traces of SCs. However, as oogenesis proceeds, the SC is restricted to the two pro-oocytes and finally to the single oocyte in region 2b. The other 15 cystocytes lose their meiotic characteristics, enter the endocycle and develop as polyploid nurse cells (Narbonne-Reveau, 2009).
During both the mitotic cycle and the meiotic cycle, it is essential that the entire genome is duplicated precisely once during the S phase. In the mitotic cycle, the licensing of the DNA occurs when Cdc6 and Cdt1/Double Parked (Dup) load the MCM2-7 complex onto the origin recognition complex (ORC) to form the prereplication complex (preRC). PreRC formation occurs in late mitosis and G1 when Cdk activity is low. At the onset of S phase, Cdk activity increases, and the preRC initiates bidirectional DNA replication. PreRC formation must be suppressed after the initiation of S phase to prevent rereplication and thus ensure that each segment of the DNA is replicated exactly once per cell cycle. Cdks play a critical role in this process by preventing reestablishment of the preRC through multiple redundant mechanisms. Thus, during the mitotic cycle the precise regulation of Cdk activity ensures that each segment of DNA is replicated once, and only once, per cell cycle (Narbonne-Reveau, 2009).
The p21cip1/p27kip1/p57kip2-like cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) specifically inhibits Cyclin E/Cdk2 complexes (de Nooij, 1996; Lane, 1996). In Drosophila, Cyclin E/Cdk2 activity is required for DNA replication during both mitotic cycles and endocycles (Knoblich, 1994; Lilly, 1996). Similar to what is observed with CKIs in other animals, Dap functions to coordinate exit from the cell cycle with terminal differentiation. Indeed, high levels of Dap are observed upon exit from the cell cycle in multiple tissues during both embryonic and larval development. Additionally, in the adult ovary, high levels of Dap prevent oocytes from entering the endocycle with the nurse cells as ovarian cysts exit the germarium in stage 1 of oogenesis (Hong, 2003). However, in addition to its well-established developmental function, recent work indicates that during developmentally programmed endocycles Dap facilitates the licensing of DNA replication origins by reinforcing low Cyclin E/Cdk2 kinase activity during the Gap phase (Hong, 2007). In dap-/- mutants, cells undergoing endocycles have reduced chromatin bound MCM2-7 complex, indicating a reduction in the density of preRCs along the chromatin. Additionally, dap-/- cells accumulate high levels of DNA damage due to the inability to complete genomic replication (Hong, 2007). Thus, during developmentally programmed endocycles Dap functions to reinforce low Cdk activity during the Gap phase (Narbonne-Reveau, 2009).
This study demonstrates that the CKI Dap promotes genomic stability during the premeiotic S phase of the Drosophila oocyte. The data indicate that Dap facilitates the licensing of DNA replication origins for the premeiotic S phase by restricting Cyclin E/Cdk2 activity during the early meiotic cycle. These studies represent the first example of a CKI regulating premeiotic S phase and genomic stability in a multicellular animal. Additionally, Dap was found to influence the timing of the mitotic/meiotic switch in ovarian cysts (Narbonne-Reveau, 2009)..
Cells in the mitotic cycle and the meiotic cycle face a similar challenge. To maintain the integrity of the genome, they must replicate their DNA once, and only once, during the S phase. In mitotic cells, this goal is accomplished, at least in part, through the precise regulation of Cdk activity throughout the cell cycle. During the mitotic cycle, Cdk activity inhibits preRC formation. This inhibitory relationship, restricts the assemble of preRCs to a short window from late mitosis to G1, when Cdk activity is low, and provides an important mechanism by which mitotic cells prevent DNA rereplication. However, the inhibitory effect of Cdk activity on preRC assembly necessitates that cells have a strictly defined period of low Cdk activity before S phase, to assemble preRCs for the next round of DNA replication. In mammals and yeast, compromising this period of low Cdk activity by overexpression G1 cyclins results in decreased replication licensing and genomic instability (Narbonne-Reveau, 2009).
One means by which cells inhibit Cdk activity is the expression of CKIs. In the mitotic cycle of budding yeast, the deletion of the CKI Sic1, which contains a Cdk inhibitor domain that is structurally conserved with the inhibitor domain present in the dap homologue p27Kip1, results in inadequate replication licensing and genomic instability due to the precocious activation of Cdks in G1. The current data strongly suggest that Dap plays a similar role in defining a critical period of low Cdk activity during the early meiotic cycle in Drosophila females (Narbonne-Reveau, 2009).
Based on these results, it is proposed that the Dap facilitates the licensing of DNA replication origins in ovarian cysts by restricting the inhibitory effects of Cyclin E/Cdk2 kinase activity on preRCs formation before premeiotic S phase. The data support the model that in the absence of Dap, ovarian cysts enter premeiotic S phase with a reduced number of licensed origins and thus fail to complete genomic replication. This hypothesis is supported by several observations. First, relative to wild-type, dap-/- ovarian cysts spend an increased proportion of their time in premeiotic S phase, as evidenced by the increased proportion of 16-cell cysts that incorporated EdU. The lengthening of premeiotic S phase is in line with the hypothesis that dap-/- ovarian cysts initiate DNA replication from a reduced number of licensed origins. Second, dap-/- ovarian cysts accumulate DNA damage during the premeiotic S phase. The accumulation of DNA damage during the premeiotic S phase is consistent with decreased preRC assembly resulting in intraorigin distances that are too large to be negotiated by DNA polymerase during a single S phase. Third, dap-/- meiotic cysts have decreased levels of the preRC component Dup/Cdt1. Moreover, genetic analysis indicates that Dup/Cdt1 levels are indeed limiting for premeiotic S phase in the dap-/- background. Specifically, it was found that reducing the dose of dup/cdt1 dramatically increases the levels of DNA damage observed in dap-/- ovarian cysts in region 2a and 2b of the germarium. In Drosophila, the levels of Dup/Cdt1 are negatively regulated by Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).
The use of the CKI Dap to restrict Cdk activity and thus promote the formation of preRCs before S phase is observed in multiple cell types beyond the oocyte. In previous work, it was found that in dap-/- mutants, cells in developmentally programmed endocycles also accumulate DNA damage and have dramatically reduced levels of Dup/Cdt1 (Hong, 2007). Thus, Dap functions to promote the accumulation of Dup/Cdt1 in multiple developmental and cell cycle contexts in Drosophila. Indeed, in select mitotic cycles removing one copy of dup/cdt1 in a dap-/- background results in DNA damage and cell death. However, in most mitotic cycles the requirement for Dap is redundant with other mechanisms that restrict Cyclin E/Cdk2 activity (Narbonne-Reveau, 2009).
Why Dap is required for preRC assembly in some cell types but not others remains unclear. However, it is interesting to note that DNA replication that occurs outside the confines of the canonical mitotic cycle, during the meiotic S phase and the S phase of developmentally programmed endocycles, is most dependent on Dap function (Hong, 2007). Thus, the increased reliance on the CKI Dap to establish a period of low Cdk activity before the onset of DNA replication may be explained by the absence of cell cycle programs that are specific to the mitotic cycle. For example, the tight transcriptional control of S phase regulators during the mitotic cycle may make the presence of Dap unnecessary for proper S phase execution. Alternatively, there may be differential regulation of the machinery that controls the regulated destruction of cyclins in the archetypical mitotic cycle versus the variant cell cycles of meiosis and the endocycle. In the future, determining why Dap plays a nonredundant role in the regulation of DNA replication during the meiotic cycle, but not the mitotic cycle, will be an important avenue of study (Narbonne-Reveau, 2009).
In addition to its role in the regulation of premeiotic S phase, this study found that dap influences the number of mitotic cyst divisions that occur before meiotic entry. In dap-/- mutants, ∼25% of ovarian cysts complete a fifth mitotic division to produce ovarian cysts with 32 cells. Similarly, mutations that compromise the degradation of the Cyclin E protein also result in production of 32-cell cysts. In line with these observations, females with reduced levels of Cyclin E produce ovarian cysts that undergo only three mitotic divisions and thus contain eight cells. Why Cyclin E/Cdk2 activity influences the timing of meiotic entry is not fully understood. However, the data suggest that the cyst division phenotype is not a direct result of reducing the number of preRCs assembled for the premeiotic S phase. Specifically, it was found that in dap-/- females reducing the dose of dup/cdt1 does not increase the number of ovarian cysts that undergo an extra division. In contrast, reducing the dose of dup/cdt1 in dap-/- females significantly enhances the meiotic DNA damage phenotype. These data strongly suggest that the extramitotic cyst division observed in dap-/- ovarian cyst is not the direct result of high CyclinE/Cdk2 activity inhibiting preRC formation (Narbonne-Reveau, 2009).
Intriguingly, Cdk2 is not the only Cdk that influences the number of ovarian cyst divisions in Drosophila females. Surprisingly, increasing the activity of the mitotic kinase Cdk1 results in the production of egg chambers with eight-cell cysts. Moreover, decreased Cdk1 activity results in ovarian cysts undergoing five mitotic divisions to produce egg chambers with 32 cells. Thus, Cdk1 and Cdk2 seem to have opposing roles in the regulation of the ovarian cysts divisions and/or meiotic entry. One of several possible explanations for these data, is that the number of ovarian cyst divisions is influenced by the amount of time cystocytes spend in a particular phase (G1, S, G2, and M) of the cell cycle. In the mitotic cycle of the Drosophila wing, there is a compensatory mechanism that ensures that changes in the length of one phase of the cell cycle result in alterations in the other phases of the cell cycle to ensure normal division rates. This compensatory mechanism is likely to be operating in multiple cell types and may account for why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions. Alternatively, Cdk1 and Cdk2 may act on truly independent pathways that have opposing roles in regulating the number of mitotic cyst divisions and/or the timing of meiotic entry. Ultimately, why Cdk1 and Cdk2 activity have opposite effects on the number of ovarian cyst divisions that occur before meiotic entry awaits the identification of essential downstream targets of these kinases (Narbonne-Reveau, 2009).
In summary, this study has defined two novel functions for a p21Cip/p27Kip1/p57Kip2-like CKI during the meiotic cycle, the regulation of the mitotic/meiotic transition and the maintenance of genomic stability during the premeiotic S phase (Narbonne-Reveau, 2009).
In higher eukaryotes, the pre-replication complex (pre-RC) component Cdt1 is the major regulator in licensing control for DNA replication. The Cul4-DDB1-based ubiquitin ligase mediates Cdt1 ubiquitylation for subsequent proteolysis. During the initiation of chorion gene amplification, Double-parked (Dup), the Drosophila ortholog of Cdt1, is restricted to chorion gene foci. This study found that Dup accumulated in nuclei in Cul4 mutant follicle cells, and the accumulation was less prominent in DDB1 (piccolo) mutant cells. Loss of Cul4 or DDB1 activity in follicle cells also compromised chorion gene amplification and induced ectopic genomic DNA replication. The focal localization of Orc2, a subunit of the origin recognition complex, is frequently absent in Cul4 mutant follicle cells. Therefore, Cul4 and DDB1 have differential functions during chorion gene amplification (Lin, 2009).
In this study, Cul4 and DDB1 mutants were isolated which were larval lethal with growth arrest in the second instar stage, similarly to previous results (Hu, 2008). It was further shown that Cul4 mutant clones in developing wing discs were defective in proliferation and had a reduced number of S-phase cells. To focus on the role of Cul4 during DNA replication and bypass the requirement of Cul4 in G1-S transition, mutant follicle cells were analyzed during gene amplification stages in follicle cells. It was shown that the Dup protein level and Orc2 focal localization are regulated by Cul4 and, differentially, by DDB1. In addition, BrdU focal patterns are defective in Cul4 and DDB1 mutant follicle cells (Lin, 2009).
Previous studies have shown the replication-dependent degradation of human and Xenopus Cdt1 by the Cul4-DDB1 E3 ligase, and the replication-coupled recruitment of the DDB1 to chromatin in Xenopus cells. This study has shown the requirement of Cul4 for the suppression of Dup protein levels during gene amplification in Drosophila follicle cells. Comparison of Dup nuclear accumulation in Cul4 and DDB1 mutant follicle cells, however, reveals substantial differences. Almost all Cul4 mutant cells, except those undergoing apoptosis, accumulated Dup in the nucleoplasm in stage 10B, consistent with Cul4 being a dedicated component of the E3 ligase in promoting Cdt1 degradation. By contrast, accumulation of Dup levels was observed in much smaller fractions of two DDB1 alleles analyzed. These analyses have not excluded the involvement of DDB1 in downregulating Dup protein levels. Compensatory or parallel Cul4-mediated Dup degradation pathways might be present in addition to the Cul4/DDB1-mediated Dup degradation. In agreement with this speculation, a recent study has shown that nuclear accumulation of cyclin D1 during the S phase promotes human Cdt1 stabilization and triggers DNA re-replication. This Cdt1 stabilization could be suppressed by the overexpression of Cul4A and Cul4B but not DDB1 or Cdt2, also an adaptor for the Cul4 ligases, implying the involvement of other adaptors in mediating the Cul4-dependent degradation of Cdt1 (Lin, 2009).
This study found that BrdU incorporation at chorion gene amplification foci was reduced or even absent in about half of Cul4G1-3 mutant follicle cells, if apoptotic cells that cannot be scored for their capability in BrdU incorporation were excluded. Similarly, 40% of Cul4G1-3 mutant cells displayed ectopic BrdU incorporation (the abnormal genomic replication group). These cells were not scored for their BrdU incorporation at focal sites because of overall strong nuclear signals. Therefore, the effect of Cul4 on the chorion gene amplification might be underestimated in this Cul4-null allele. Using the Cul4 hypomorphic allele KG02900 in which both the fractions of apoptotic cells and abnormally BrdU-incorporated cells were reduced, the combined percentages for reduced and absent BrdU incorporation combined at chorion gene amplification foci reached more than 70%. DDB15-1 mutant follicle cells also displayed a severe phenotype in the BrdU incorporation assay. When apoptotic cells were disregarded, cells with a reduction or absence of BrdU incorporation accounted for more than 60% of DDB15-1 mutant cells. Therefore, these BrdU incorporation analyses lend support to the notion that certain processes in chorion gene amplification require both Cul4 and DDB1 (Lin, 2009).
These BrdU foci represent DNA amplification of chorion genes within 100 kb of origins, and the phenotype of absence or reduction in BrdU incorporation could reflect a failure in the initiation of DNA replication, reduced processivity in DNA synthesis or fewer rounds of gene amplification. To further support the idea that Cul4 is involved in gene amplification, advantage was taken of the dominant-negative Cul4KR mutant in which the neddylation site has been mutated. Acute expression of Cul4KR suppressed BrdU incorporation in almost all follicle cells. When assayed by quantification PCR, gene amplification at chorion foci was strongly suppressed, supporting a role of Cul4 in the chorion gene amplification process (Lin, 2009).
Abnormal genomic replication, as inferred from ectopic BrdU incorporation, was observed in Cul4 mutant follicle cells in both Cul4 mutant alleles tested. The percentage of cells with such a defect was reduced in the cells with the hypomorphic mutant allele, indicating that the low level of Cul4 activity partially suppresses this defect. Abnormal genomic replication was also detected in DDB1 mutant follicle cells with a lower frequency than in Cul4-null mutants. The phenotype of ectopic genomic replication is less likely to be a retarded developmental process in the previous endocycle stage, because these cells with ectopic BrdU signals show normal DNA contents as estimated by Hoechst staining. Ectopic genomic replication might require some prerequisite steps in DNA replication, such as Orc2 localization at replication origins, which is defective in Cul4 mutant cells, thus blocking abnormal DNA replication throughout the genome (Lin, 2009).
The localization of Orc2, a component of the pre-RC, at chorion gene foci was examined during gene amplification. Mutations in Cul4 caused reduced or no Orc2 localization at gene amplification foci, a prominent phenotype in both G1-3 (59%) and KG02900 alleles (60%) when apoptotic cells were discounted. Failure of proper Orc2 focal localization might represent defects in the initial loading of Orc2 or the maintenance of Orc2 localization at amplification foci. Such Orc2 localization defects were not prominent in DDB1 mutant follicle cells (Lin, 2009).
Consequently, defective Orc2 localization at regular gene amplification foci might evoke ectopic genomic replication in Cul4 mutant follicle cells. Upon co-labeling for Orc2 and BrdU in Cul4 mutant cells, the absence of or reduction in Orc2 signals was found in conjunction with a reduction in BrdU incorporation at gene amplification loci or with abnormal BrdU incorporation throughout the genome. In some cells, normal Orc2 loading was accompanied with abnormal BrdU incorporation. The decoupling of both phenotypes therefore suggests that Cul4 functions distinctively in Orc2 localization and in suppression of abnormal BrdU incorporation during chorion gene amplification (Lin, 2009).
Many interesting questions remain to be answered regarding how genetic loci are selected for amplification, how the pre-RC is assembled only in specific loci and how other genomic regions are kept silent. Previous evidence suggests that high transcriptional activity of specific loci controls replication origin firing during the gene amplification stage. Mutants for transcription factors such as E2f2, Dp, Rbf, Myb and Mip130 show increased mRNA and protein levels of replication factors, such as components of the Orc and MCM complexes, and ectopic genomic replication during the gene amplification stage. Mutant follicle cells for Cul4 displayed both Orc2 localization defects and abnormal genomic replication, implying that Cul4 is probably involved in both processes by modulating the transcriptional activity in DNA replication. Interestingly, a recent study suggests that Cul4 targets degradation of the transcriptional activator E2F1 during S phase. However, Drosophila E2F1 proteins became abundant in the nucleus and rich at ACE3 origin DNA during gene amplification. How this developmental regulation of E2F is involved in Cul4 activity needs further investigation. Some studies suggest that Cul4 functions in histone modification and heterochromatin maintenance. It is speculated that the Cul4 E3 complex also functions in regulating Orc2 origin localization through a local remodeling of the chromatin structure on ACE3 and Ori-β (Lin, 2009).
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).
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