BIOLOGICAL OVERVIEW

In order to locate disc proliferation abnormal (dpa) in what might be described as a roadmap for the process of DNA replication, one first must look at the other points on the map.

These are fairly well known (see the DNA replication site), and include DNA polymerases, a DNA ligase, a single stranded DNA binding protein, a processivity factor called Proliferating cell nuclear antigen, and a Tropoisomerase. But what proteins are needed to trigger initiation of DNA replication, and how do they intersect the other proteins and processes involved? Two sets of factors are required. One factor, called the origin recognition complex (ORC), recognizes origins of replications, and signals to the DNA replication apparatus where to carry out its function. For more information about the ORC in Drosophila, see the ORC2 site. Another factor 'licenses' DNA replication, that is, it gives the go ahead for initiation of DNA replication. Information from other organisms, particularly yeast and Xenopus, has allowed the identification of a group of genes that code for called minichromosome maintenance (MCM) proteins that serve to license DNA replication. MCM proteins regulate initiation of DNA synthesis in a cell cycle dependent fashion.

Two MCM genes are known in Drosophila, Minichromosome maintenance 2 and disc proliferation abnormal. Mutations in either gene cause a similar phenotype: cells of imaginal discs are unable to replicate DNA and therefore cells of imaginal discs are unable to multiply. Apparently enough MCM proteins are available from the mother to carry the embryo through the initial stages of development. DmMCM2 mutants exhibit central nervous system defects, and mutations in both genes cause pupal lethality (Feger, 1995 and Treisman, 1995). The following is a summary of the history and biochemisty of DNA proliferation licensing factor.

The first signal for initiation of DNA replicaton involves replication licensing factor (RLF); this licenses replication origins by putting them into an initiation-competent state. The second signal, S-phase promoting factor (SPF), induces licensed origins to initiate, and in doing so removes the license. RLF was first characterized in Xenopus as a component of cell-free extracts supporting chromosomal DNA replication in vitro. In this system, RLF (the license) and SPF (the initiation signal) are prevented from acting at the same time in two different ways. First, RLF cannot cross the nuclear envelope; it can only license DNA when the nuclear envelop is broken down in mitosis. In contrast, SPF can only initiate DNA replication on licensed DNA within an intact nucleus. Second, the spatial separation provided by the nuclear envelope is reinforced by a temporal separation, as both activities are periodic in the cell cycle, that is, they are subject to regulation by cyclins. RLF is activated abruptly during the metaphase-anaphase transition and decays during interphase, while SPF activity can only be detected during interphase.

RLF of Xenopus can be separated into two essential components, RLF-M and RLF-B, both of which are required for licensing. RLF-M, a fraction containing members of the minichromosome maintenance family, associates with chromatin prior to replication but is removed during replication. RLF-M's reassociation with chromatin requires passage through mitosis. RLF-M requires RLF-B ( an as yet uncharacterized fraction) for binding RLF-M to DNA. Apparently RLF binds to origins of replication, but the basis for this binding has not yet been characterized (Chong, 1996 and references).

All six MCM family members (MCM2, 3, 4, 5, 6 and 7) are homologous proteins forming six MCM evolutionary groups. There is only a single gene from any one group in each species. RLF-M complex in Xenopus consists of three or more MCM proteins including MCM2, 3 and 5. In human cells MCM4 is found associated with MCM2, 3 and 7. It is possible that complexes containing different combinations of MCM proteins might be required for initiation of different classes of replication origin. Unlike in S. cerevisiae, where there appears to be cell-cycle-regulated entry of MCM proteins into the nucleus, MCM proteins in mammalian cells and Drosophila are constitutively nuclear. Despite this, mammalian MCMs show intranuclear cell-cycle variation: they are displaced from chromatin during S phase, and do not re-bind until progression into mitosis. The inability of displaced MCMs to re-bind chromatin is most easily explained by a lack of RLF-B activity (the second component of the licensing system) in the nucleus (Chong, 1996 and references).

A candidate for RLF-B type activity is the Cdc6 protein of S. cerevisiae. Cdc6 protein is synthesized during mitosis. in vivo footprinting techniques have been used to examine protein occupancy of the DNA at the origin core site throughout the budding yeast cell cycle. The protective pattern in G2 cells closely resembles that of the purified Origin recognition complex with DNA; late in mitosis (in anaphase) this protection increases by about 50 base pairs. In striking analogy to the behavior of the MCM complex, this extended footprinting is lost during S phase. This preinitiation footprinting and its half-life are dependent upon the action of Cdc6 protein. Thus Cdc6 protein could be tethering the MCM complex to the origin recognition complex (Botchan, 1996 and references).

What are the functions of the MCM proteins? Each protein has a molecular weight of 80-120kDA and a highly conserved central region. This central region contains a predicted consensus sequence for DNA-dependent ATPases and shows some homology to DNA helicases. As DNA unwinding is expected to be required at replication origins, it is attractive to think that this is the function of MCM proteins (Chong, 1996).

Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells

The MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, it was of interest to determining whether the entire cellular content was necessary for DNA replication in vivo. A systematic depletion of the MCM proteins was performed in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95-99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. Depletion of MCM8 caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5. These studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells (Crevel, 2007).

Although this study could demonstrate specificity of the RNAi depletions, co-instability was observed for certain combinations of MCM proteins. Some of observations can be explained based on the composition of reported MCM sub-complexes. Therefore, a reduction in MCM5 when MCM3 is targeted, and MCM4 when MCM6 and MCM7 are targeted might be related to loss of stability of the MCM3/5 and MCM4/6/7 complexes. However this is not a complete explanation since MCM3 stability is unaffected by MCM5 depletion and MCM6/7 are not affected by depletion of other MCM4/6/7 components. In addition, MCM2, which is affected by MCM6 depletion, is not thought to be a component of either complex. The co-reductions cannot be rationalized based on models proposed for the structure of the MCM hexameric complex. Although the basis for the co-reductions is not understood, it was shown that they did not occur via the proteosome pathway since treatment with proteosome inhibitors had no effect. Co-instability of MCM proteins has been reported in other systems, but the reported combinations differ from those observed in Drosophila (Crevel, 2007).

Secondly the data suggest that a dramatic reduction in the level of MCM2-6 and 8 in vivo in Drosophila S2 cells has little apparent effect on cell survival and DNA replication. This therefore suggests that the MCM paradox - originally observed in Xenopus cell free extracts - can also be observed in vivo for Drosophila. Cell viability has also previously been shown to be unaffected for MCM2 and 5 depletions in Drosophila Kc cells. Whether the same effects will also be seen in other higher eukaryotes is unclear and in fact it has been reported that human cells cannot traverse S if MCM4 is depleted (Ekholm-Reed, 2004). Since it is unlikely that the lack of replication effects on depletion of MCM2-6 is due to a different role for the MCM complex in Drosophila, two other possibilities are suggested. Firstly, consistent with what has been suggested for Xenopus it might be that under normal circumstances most of the MCM protein in cells is redundant. It is estimated that there are 50-100 MCM complexes per origin (assuming origin spacing of 40-100 kb) in S2 cells. Therefore even cells which have lost 99% of a specific MCM should have enough protein to ensure that most origins have one MCM complex. A single MCM complex per origin may therefore be sufficient to allow a full complement of activated replication forks as measured by PCNA loading. In this case the results support proposed MCM mechanisms involving single or double hexamers rather than those that require bulk chromatin loading of MCMs. Perhaps in support of this suggestion no effects are seen of the MCM depletion on cdc45 chromatin loading. Cdc45 has been suggested to form an active component of the replicative helicase complex with GINS and MCM proteins (Moyer, 2006). It is therefore possible that despite the drastic reduction in the total number of MCM complexes in the dsRNA treated S2 cells the total number of active helicases has not altered (Crevel, 2007).

The second possibility is that MCM loss is compensated for by other proteins. Whether MCM8 could perform this function was investigated. The decrease in PCNA loading observed on depletion of MCM8 suggests that Drosophila MCM8 does play a role in replication. From these data the exact nature of its role is unclear, however the lack of an effect of the depletion on cdc45 loading suggests that unlike the MCM2-7 proteins it is unlikely to be required for the loading of downstream initiation factors. In addition MCM8 cannot be the MCM2-6 compensating protein since co-depletion of MCM5 and MCM8 does not synergistically affect cell viability or DNA replication (Crevel, 2007).

Finally the differences observed between depletion of MCM7 and MCM2-6 suggest that not all MCM proteins are equivalent. The mechanism behind this differential behaviour is not clear. Although a complex of MCM4/6/7 has been shown to have helicase activity, there is no evidence that MCM7 acts independently. Therefore MCM7 may have additional cellular functions. A role for MCM7 as a damage sensor via the ATR pathway has been suggested by work in human and S. cerevisiae cells where depletion or mutation of MCM7 produces cells defective in the UV-induced S-phase checkpoint. In Xenopus extracts MCM7 has also been shown to bind to the Rb protein leading to a brake on DNA replication. It is possible that the MCM7 effect that was observed is related to a failure of a negative control. This could lead to more significant damage which activates other checkpoints to cause the S phase stop. How this might be related to the roles of the S.cerevisiae and human MCM7 protein in the UV checkpoint is unclear since RNAi depletion of human MCM7 was not reported to show this effect. The level of MCM7 protein remaining after depletion is significantly higher in human than Drosophila cells, however less efficient depletion of Drosophila MCM7 has been seen to produce the same effect. Alternatively in addition to acting as a negative regulator of replication, MCM7 may have a positive regulatory effect on replication. In either case the effect is likely to involve MCM7 directly, rather than occurring as a secondary effect of a replication defect, since similar effects are not observed for other MCM proteins (Crevel, 2007).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 866

Structural Domains

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the DEAD and DEAH box families ATP-dependent helicases signatures.

The DPA protein is 45% identical to fisson yeast cdc21 [an MCM4 homolog](45% identity), the budding yeast CDC54 [another MCM4 homolog] (44%) , CDC46 [an MCM5 homolog](34%), MCM3 (31%) and mammalian P1 [an MCM3 homolog] (31%) (Feger, 1995 and Chong, 1996). Based on sequence similarity, DPA can be assigned to the CDC21/CDC54/MCM4 branch of the MCM gene family. The highest sequence similarity exhibited between members of this family of genes is found in the middle domain. The N-terminal domain of DPA contains a putative Zn finger. The Zinc finger motif, conserved in cdc21, CDC54 and MCM2, has been shown to be essential for MCM2 function. DPA contains five potential phosphorylation sites for cdc2 protein kinase at its N-terminus and a sixth cdc2 phosporylation site following the Zinc finger motif. CDC21, CDC54 and MCM2 also contain potential cdc2 phosphorylation sites, although the number and location of these sites differ in each case (Feger, 1995).

date revised: 7 March 98

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