BIOLOGICAL OVERVIEW

double parked (dup) is essential for DNA replication and belongs to a new family of replication proteins conserved from Schizosaccharomyces pombe to humans. Strong mutations in dup cause embryonic lethality, preceded by a failure to undergo S phase during the postblastoderm divisions. dup is required also for DNA replication in the adult ovary, establishing that dup is needed for DNA replication at multiple stages of development. Strikingly, Dup protein colocalizes with the origin recognition complex (see Orc2) to specific sites in the ovarian follicle cells. This suggests that Dup plays a direct role in DNA replication. The dup transcript is cell cycle regulated and is under the control of E2F and Cyclin E. Interestingly, dup mutant embryos fail both to downregulate S phase genes and to engage a checkpoint preventing mitosis until completion of S phase. This could be either because these events depend on progression of S phase beyond the point blocked in the dup mutants or because Dup is needed directly for these feedback mechanisms (Whittaker, 2000).

dup mutants are defective in DNA replication both in embryogenesis and in oogenesis. To analyze DNA replication in dup mutants, embryos were isolated from females heterozygous for dup^a1 that had been crossed to heterozygous males and pulse labeled with bromodeoxyuridine (BrdU). Homozygous mutant embryos were distinguished from heterozygous embryos by using a marked balancer chromosome. In the dup mutants embryonic DNA replication appears to be normal through S phase of cycle 15. This is most likely because maternal pools of Dup protein suffice for the earlier embryonic replication cycles. In contrast, BrdU incorporation is not detectable in cycle 16. The block in replication in dup^a1/dup^a1 homozygous mutant embryos occurs early in S phase, because no BrdU incorporation is seen in the nuclei (Whittaker, 2000).

In addition to its role in embryogenesis, the viable fs(2)PA77 mutation demonstrates that dup is necessary for DNA replication in adult tissues. Homozygous mutant fs(2)PA77 females lay eggs with thin egg shells, and chorion amplification levels are decreased. Follicle cell genomic replication and amplification can be visualized directly by BrdU labeling of ovaries. Between stage 9 and early stage 10A genomic replication ceases, and BrdU incorporation is no longer detectable throughout the nucleus. In stage 10B, BrdU is observed at four specific foci that are sites of amplification. Previous BrdU incorporation studies have shown that chorion amplification is decreased uniformly in the follicle cells of fs(2)PA77 homozygous mutant females (Calvi, 1998). Similarly, amplification in only a few stage 10B eggchambers is observed. Although incorporated BrdU is detectable in stage 11 eggchambers, the size of the foci is smaller, and this is indicative of reduced levels of amplification (Calvi, 1998). In addition to the amplification defects, genomic replication persists longer than normal in the fs(2)PA77 homozygous mutant females, since genomic replication is still seen in some follicle cells in late stage 10A eggchambers (Whittaker, 2000).

Occasionally fs(2)PA77/dup^a1 female flies are recovered. BrdU incorporation in ovaries from these flies has been examined and incorporation into the follicle cell genome is found to be reduced relative to wild type. This reduction is most apparent in stage 9. Moreover, no BrdU incorporation is detected at the chorion loci at any stage. These results indicate that genomic replication is decreased in dup mutants and that the initiation of amplification requires functional dup. Thus, dup is essential for replication in multiple stages of development and is required for genomic replication in both mitotic and endo cycles and also for amplification (Whittaker, 2000).

The subnuclear localization pattern of Dup at stages 10B and 11 is strikingly similar to that seen for Orc2 and Orc1, which have been shown to localize to the sites of chorion gene amplification at these stages. At stage 10B, Dup and Orc2 colocalize in the follicle cells, although the Dup staining appears more diffuse than that of Orc2. Two interesting differences are seen in the localization pattern of Orc2 and Dup. (1) At stage 10A, Orc2 has already localized to subnuclear foci. Dup, in contrast, is still diffusely localized in the nucleus. (2) Orc2 is not seen to localize to origins after stage 11. Interestingly, Dup is undiminished and remains detectable at subnuclear foci until stage 13, indicating that it may function during elongation. Alternatively, Dup could have no role in elongation but is not cleared from chromatin until after elongation has been completed (Whittaker, 2000).

Because ORC is localized specifically before Dup, a test was performed to see whether ORC function is a prerequisite for Dup localization at replication foci. There is a female-sterile mutation in the orc2 gene, fs(2)293, that is defective in amplification. The effect of the orc2 mutation on ORC localization has been examined. In fs(2)293 mutants, the amount of Orc2 is greatly reduced or undetectable at specific foci during amplification. Dup is not localized to specific foci in this mutant in many follicle cell nuclei. In other follicle cells, Dup can be seen at one focus, but the levels are greatly reduced. This suggests that Orc2 is required for Dup localization. Interestingly, in some nuclei, high nuclear levels of Dup are observed. The levels are higher than those seen during stages 7-10A. Additionally, DAPI staining of the nuclei doesn't indicate that morphology of the cells is altered. This may suggest that Orc2 is required either indirectly through its requirement for replication, or directly, to prevent nuclear accumulation of Dup. It may be that Dup enters the nucleus independently of ORC, but that ORC is required to localize it to the amplifying regions (Whittaker, 2000).

To determine if Dup is necessary for continued ORC localization, Orc2 localization was examined in the fs(2)PA77 mutant. The shape and number of Orc2 foci is normal in the mutant follicle cells, but the size is smaller. One interpretation of these results is that Dup plays a role in loading or maintaining Orc2 at the amplification origins. Because the shape and number of foci seen are appropriate for the stages examined, it was thought to be more likely that the decrease in levels of Orc2 localization results from lower levels of DNA replication, causing decreased copy number of the origins. dup is not required for normal levels of the MCM (see Disc proliferation abnormal) proteins in the nuclei (Whittaker, 2000).

If S phase is blocked during the postblastoderm divisions by treatment with aphidocolin, the cell cycle arrests and mitosis does not occur. In yeast it has been demonstrated as well that arrest in S phase stops the cycle and blocks mitosis. In contrast, in yeast, mutation of the Cdc18/Cdc6 protein required to initiate DNA replication does not block mitosis even though DNA replication does not take place. Therefore, it was of interest to test whether mitosis occurs after the S phase block in dup mutant embryos to determine whether this checkpoint is functional in dup mutants. The histone H3 phosphoH3 epitope (PH3) present on condensed chromosomes was used to test for the presence of mitotic cells in dup mutant embryos. Normally, in embryos that have completed cycle 16, PH3 antibodies label mitotic cells solely in the CNS and the PNS. No labeling is seen in the G0 epidermal cells or in the endo cycling cells. In contrast, in dup^a1/dup^a1, dup^a1/dup^a3, and dup^a3/dup^a3 mutant embryos many cells in the epidermis label with anti-phospho H3 antibodies. There are also some labeled cells in the gut in addition to the CNS and PNS. This defect is first manifest at mitosis 16, after the block in S phase is observed. The mitotic block occurs after the failure of DNA replication because closer examination of the chromosomes show that they are 2N. In addition, the number of nuclei in stage 11 embryos indicates that mitosis 15 takes place in dup mutant embryos. The number of nuclei within each segment of the mutant embryos is about half that of wild type, implying failure of the last division of embryogenesis (mitosis 16) but not the last two divisions. Although the possibility that in some cells mitosis 15 does not occur cannot be excluded, it does take place in the majority of the cells (Whittaker, 2000).

The labeled cells were blocked in an abnormal mitosis 16 with unreplicated chromosomes. The chromosomes appear over-condensed and in some cells, fragmented. No telophase figures were detected, and thus the mutant cells do not complete mitosis. After stage 12, as development progresses, the number of anti-phospho H3 labeled cells decreases. Because no indication that these cells completed mitosis was seen, this may be due to cell death. However, it is also possible that these cells abort mitosis and return to G0 or G1 phase. Thus, despite the failure to undergo S phase, cells enter mitosis in dup mutant embryos (Whittaker, 2000).

The dup mutant embryos are defective in another cell cycle process in that they fail to downregulate transcripts that were induced at the G1-S transition. This phenotype is the basis by which mutations in dup were originally isolated in a screen designed to recover mutations in G1-S transcription. PCNA transcripts remain at high levels after S phase 16 in dup mutants, rather than being downregulated. It was of interest to determine if dup mutations affect other S phase genes similarly. To test this, in situ hybridization with cyclin E and RNR2 riboprobes was done on dup mutant embryos. In all four alleles of dup, S phase transcripts fail to be down regulated in both the epidermis and the endo cycling cells. In older stage 14 embryos the level of transcript appears to decrease. Similar to staining with anti-phospho H3, this could be due to cell death occurring in the epidermis or cells reentering G0 or G1 phase. This failure to downregulate S phase transcripts is not the result of mitotic arrest in dup mutants, because pimples mutant embryos arrested at metaphase of cycle 16 downregulate transcripts properly (Whittaker, 2000). This transcriptional phenotype may indicate that dup plays a direct role in transcriptional regulation. Alternatively, the failure to down regulate S phase transcripts may simply result from a failure to complete S phase. The latter interpretation is favored for two reasons: (1) the replication phenotype precedes that of the transcription phenotype and (2) the female-sterile mutation in dup does not affect the levels of PCNA transcripts in the ovary (Whittaker, 2000).

Thus Dup is a member of a conserved family of replication proteins with homologs in organisms ranging from S. pombe to humans. Dup colocalizes with Orc2 to chorion amplification foci, suggesting that it is part of the replication machinery. The dup mutants are striking also because they lack the checkpoint that would normally prevent mitosis if replication were incomplete. This may be either because Dup is part of this regulatory pathway or because S phase does not proceed sufficiently far in the mutants for the cells to sense that replication is incomplete. Furthermore, dup mutants fail to downregulate S phase transcripts on the addition of the first G1 phase in embryogenesis, suggesting that completion of S or passage through a certain point in S phase may be required for this downregulation (Whittaker, 2000).

These results show that Dup is essential for replication at multiple stages of development: during the post blastoderm mitotic cycles (the endocycles), and the amplification that occurs during oogenesis. Dup is probably required for S phase in the early embryonic cycles as well, as the maternal pools of protein could account for why a replication defect is not detected until cycle 16. The mutant phenotypes do not distinguish whether Dup acts in initiation of DNA replication, elongation of replication forks, or both. Two results suggest that Dup acts after ORC in replication: (1) Orc2 is still localized in dup mutants; (2) Dup localization to foci does not become apparent until after Orc2 is localized. The fact that no BrdU incorporation is detectable in strong dup mutants suggests an early block in replication and a role for Dup in initiation. Because Dup foci persist longer than Orc2 foci and Dup remains localized during stages when amplification elongation rather than initiation is occurring, Dup may function in elongation as well. This does not exclude a role of Dup in initiation, since the MCM proteins are part of the preinitiation complex at origins but also appear to move with the replication fork. Further experiments will be required to determine whether Dup is present both at moving replication forks as well as the amplification origins (Whittaker, 2000).

The similarity between Dup and the S. pombe protein Cdt1 suggests a model for how Dup functions in replication. Mutations in cdt1 block DNA replication initiation (Hofmann, 1994). Recent experiments show that Cdt1 accumulates in the nucleus in G1 and is a component of the prereplicative complex (Nishitani, 2000). Cdt1 protein is in a complex with the Cdc18 protein, the S. pombe homolog of the Cdc6 initiator protein. In addition, loading of MCM proteins onto chromatin is blocked in cdt1 mutants (Nishitani, 2000). An ortholog to Cdt1 has been identified in Xenopus and shown to be required for DNA replication in vitro to load MCM proteins (Maiorano, 2000). Consistent with the results on Dup localization, Xenopus Cdt1 association with chromatin is dependent on ORC. However, in Xenopus extracts Cdt1 dissociates from chromatin after the initiation of DNA replication (Maiorano, 2000). By analogy to Cdt1, Dup may be needed in conjunction with Cdc6/Cdc18 to help load MCM proteins onto the origins, but also to maintain them at the replication fork. Because the MCM proteins are not specifically localized during amplification in the follicle cells, no effect of the dup mutations on MCM localization could be detected. Therefore, tests of this model will require biochemical approaches (Whittaker, 2000).

Interestingly, no Dup homolog has been found in S. cerevisiae. Dup may be needed for replication initiation in eukaryotes that have more complex origins of replication, and hence are not present in S. cerevisiae. Since sites of initiation in these eukaryotes are less specific than in S. cerevisiae, it is possible that Dup plays a role in helping to direct MCMs to the appropriate origins. This would be especially useful in higher eukaryotes where origin usage varies by cell type and time in development. Alternatively, S. cerevisiae may contain an analogous protein that is sufficiently diverged so as to have not been recognized in similarity searches. The MCM10 protein is needed to load MCM proteins into the S. cerevisiae prereplicative complex, so MCM10 could perform the same function as Cdt1/Dup in metazoans (Whittaker, 2000).

The dup mutations uncouple the dependency of mitosis upon completion of S phase. This may be because dup is part of the machinery that implements this checkpoint. However, precedents in yeast support the hypothesis that the failure to engage this checkpoint in dup mutants is a consequence of replication not initiating and not producing stalled intermediates that can be recognized. In both S. cerevisiae and S. pombe, mutations that block initiation of DNA replication do not block progression into mitosis, whereas arrest during elongation stages of DNA replication does. Therefore, if Dup is indeed critical for initiation of DNA replication, the absence of DNA synthesis may account for the checkpoint failure without any direct involvement of Dup in the machinery that signals the checkpoint (Whittaker, 2000 and references therein).

Mutations in dup also reveal a likely requirement for the initiation of S phase for the subsequent downregulation of S phase transcripts. Interestingly, mutations in string that block in G2 phase and prevent S phases 15 and 16 do not prevent downregulation of transcripts, nor does arrest in metaphase by mutations in three rows or pimples. Therefore, the failure to downregulate the level of S phase transcripts in dup mutants is not the consequence of a simple block in the cell cycle or complete prevention of S phase. It is logical that a cell would not downregulate the transcripts encoding products required for DNA replication until initiation had occurred, possibly reflecting a strategy to drive S phase to completion by maintaining high levels of the replication machinery. Interestingly, in S. pombe, cdc18 transcript is not downregulated if S phase is blocked with hydroxyurea, indicating that downregulation may require the completion, not merely the initiation, of DNA replication (Whittaker, 2000 and references therein).

GENE STRUCTURE

cDNA clone length - 2761

Bases in 5' UTR - 211

Bases in 3' UTR - 318

PROTEIN STRUCTURE

Amino Acids - 743

Structural Domains

Blast searches with the Dup sequence reveal that it is a member of a conserved family of proteins. The carboxy-terminal end of Dup shares very high similarity to a fully sequenced human cDNA the function of which has not been reported. The human cDNA is 34% identical and has 52% conservative changes compared to the carboxy-terminal 446 amino acids of Dup. The human cDNA sequence has been reported to be identical to a sequence 3' to the human adenine phosphoribosal transferase gene, making it likely that human Dup maps to this region on chromosome 16. Dup also shows high homology to several partially sequenced mouse ESTs. One of these shows 42% identity and 63% positives when compared to the carboxy-terminal end of Dup. There are predicted protein orthologs to Dup protein in Arabidopsis thaliana and Caenorhabditis elegans (22% identity and 36% positives and 23% identity and 39% positives, respectively). Interestingly, Dup is also related to S. pombe Cdt1 (20% identity and 37% positives), a protein known to be required for replication initiation (Hofmann, 1994). Alignments of Dup, Cdt1, and the human, C. elegans and A. thaliana orthologs show that these proteins share several conserved motifs, making it likely that they represent members of the same family of proteins. Arginine 342, which is changed to a cysteine in the fs(2)PA77 allele is conserved in humans, mouse, and A. thaliana, consistent with it playing an essential role in the function of Dup. The amino acid sequence of Dup does not reveal predicted biochemical activities (Whittaker, 2000).

date revised: 30 October 2000

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