InteractiveFly: GeneBrief

Minichromosome maintenance 5: Biological Overview | References

Members of the minichromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. This study provides a genetic dissection of the mcm5 gene in Drosophila that demonstrates an unexpected function for this protein. First, it is shown that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, a viable and fertile allele of mcm5 (denoted mcm5^A7) was recovered that specifically impairs the meiotic recombination process. The decrease in recombination observed in females homozygous for mcm5^A7 is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), but rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5^A7 are fully proficient in somatic DNA repair. These results strengthen the observation that members of the prereplicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway (Lake, 2007).

MCM proteins were first identified for having a role in the maintenance of plasmids (minichromosomes) in proliferating cells of Saccharomyces cerevisiae. In part, they function to ensure the faithful transmission of the genome from one generation to the next; however, in addition to a critical role in DNA replication, members of this family are now thought to be multifunctional and also play roles in transcription, cohesion, condensation, and recombination (Forsburg, 2004; Lake, 2007 and references therein).

All MCM members belong to the AAA+ ATPase family, which has a distinct ATPase domain that spans ~200 bases. This domain, referred to as the MCM box, consists of a Walker A ATPase motif, a Walker B ATPase motif, and an arginine finger motif (R-finger). Conserved sequences within the Walker B motif (IDEFDKM) and R-finger (SRDF) define the MCM family. Six of these members are conserved in all eukaryotes and form a heterohexameric complex known as Mcm2-7, which has been studied extensively for its role in DNA replication. Mcm2-7 is required for licensing and initiating origins of replication, and it acts during elongation as a helicase at the replication forks. Because of this function and studies in yeast, Arabidopsis and Drosophila, members of the Mcm2-7 complex, are thought to be essential. In addition, two other MCM family members, Mcm8 and Mcm9, have recently been identified and are thought to be a distinct subgroup of MCM proteins (Maiorano, 2006). Mcm8 has been reported in vertebrates and Drosophila, but not in fungi and nematodes, and although it retains some sequence similarities in the Walker B and R-finger, its Walker A ATPase motif contains sequences more like the canonical ATPases. Mcm9 is also found in similar organisms with the exception that it is missing in Drosophila, and it is unique to the family in that it lacks the carboxy-terminal ATPase domain including the Walker B motif (Lake, 2007 and references therein).

Recently, studies have indicated that in addition to the role in DNA replication, certain members within the Mcm2-7 complex, as well as other MCM family members, have functions outside of DNA replication (Forsburg, 2004). Specifically, some of these functions include a role in transcriptional activation, chromosome condensation, cohesion, and recombination. The existence of multiple functions is consistent with studies in yeast, which showed that MCM proteins are far more abundant than would likely be required for the number of replication origins that exist, and this abundance cannot explain the fact that slight decreases in amounts of MCM proteins lead to the inability to complete S-phase and progress through the cell cycle. Moreover, in addition to the heterohexameric Mcm2-7 complex, subcomplexes of MCM family members have been identified, which fuel the speculation that these complexes could be functionally distinct subgroups that possess functions beyond those involved in DNA replication (Lake, 2007 and references therein).

Limited functional studies have been done on the Drosophila orthologs of the Mcm2-7 complex. Although genes for each of these members have been identified in Drosophila, only mcm2, mcm4 (Feger, 1995), and mcm6 (Schwed, 2002) have been shown to be required for mitotic DNA replication. Null alleles of each inhibit proliferation of cells of the central nervous system (CNS) and imaginal discs, which leads to a reduction in brain size and lack of discs within the developing larvae. These larvae begin pupariation but never develop into adults. In addition to a role in mitotic DNA replication, two other functions for mcm6 have been identified that were not observed in mcm2 or mcm4 mutants. Mcm6 is required for endo-reduplication which is a process of reoccurring rounds of DNA replication in the absence of cell division that occurs within the developing larvae and is responsible for most of the larval growth and is also required for chorion gene amplification (Lake, 2007).

Until now, there have been no genetic studies that analyzed the roles of mcm5 in Drosophila. Although it is speculated that mcm5 is required for DNA replication in Drosophila, specific functions in this process as well as other functions it may have remain unknown. The fortuitous identification of an allele of mcm5 in a screen for new meiotic mutants stimulated a thorough genetic dissection of this gene to determine the functions of mcm5 in Drosophila (Lake, 2007).

This study shows that the mcm5 locus is essential, in that homo- or hemizygotes for a null mutation in mcm5 die prior to eclosion. They do, however, survive to third instar larvae with rudimentary imaginal discs and small brains, suggesting a defect in facilitating mitotic DNA replication. This defect in mitotically dividing cells does not extend to endo-reduplicating tissues, since the highly polytene chromosomes of the salivary gland appear normal. These findings are similar to what has been identified for mutants in mcm2 and mcm4, but differ from findings in mcm6, which is also considered essential for endo-reduplication (Lake, 2007).

In addition to the null allele of mcm5, an EMS-induced allele was identified that is not required for the essential functions of Mcm5, which is to say that homo- and hemizygotes for this mutant are viable and fertile, but rather has a function in the meiotic recombination pathway. The decrease in recombination is not due to a failure to form either synaptonemal complex or double-strand breaks (DSBs) or to a general inability to repair DSBs that are induced by DNA damaging agents in somatic cells. This observation suggests that a residue or domain in the Drosophila mcm5 gene has been identified that is specifically required for meiotic recombination (Lake, 2007).

The genetic analysis of the mcm5 gene in this study allowed determination that the mcm5 gene product has two distinguishable functions in Drosophila: an essential function in mitosis and a specialized function in the meiotic recombination pathway. Like other MCM family members in Drosophila, mcm5 has a phenotype consistent with a role in mitotic DNA replication. However, it is not required for the processive DNA replication cycles that occur during endo-reduplication in the salivary glands of the developing embryo (Lake, 2007).

A function of Mcm5 has been identified in the maturation of DSBs into crossovers; this meiotic function is separable from the role of Mcm5 in mitotic replication. A residue or domain in Mcm5 has been identified that is specifically required for this meiotic function. The observation that mcm5^A7 homozygotes and mcm5^A7/Df heterozygotes demonstrate similar levels of X chromosome nondisjunction argues that the mutant constitutes a null allele with respect to the role of this protein in meiotic recombination, while the fact that this mutant so strongly affects meiosis without affecting viability demonstrates that the mcm5^A7 mutant is a clear separation-of-function allele in terms of the role of the Mcm5 protein in mitosis and meiosis (Lake, 2007).

Recombination-deficient mutants in Drosophila can coarsely be grouped into four classes: those like mei-W68 (which encodes the fly homolog of SPO11) that block the formation of DSBs, those like mei-9 and mus312 that are involved in the resolution of recombination intermediates, those like spn-A and spn-B that are involved in the repair of DSBs, and a class of mutants, often referred to as precondition mutants, that appear to simply alter the probability that DSBs will be processed into crossover events. Precondition mutants are characterized by the fact that they not only decrease the total number of exchange events, but also alter the mechanisms that normally control the distribution of exchanges, such that exchanges occur more commonly in proximal regions than in distal regions. They also usually ablate crossover interference, the process that serves to distribute crossover events along the arms of chromosomes. The mcm5^A7 mutant, as well as mutants in the mei-218, rec/mcm8 genes, are all precondition mutants. The three genes defined by these mutants encode either bona fide MCM proteins (Mcm5 or Mcm8) or a protein with a MCM domain (Mei-218) (Lake, 2007).

This study has shown that DSBs are both created and disappear with normal kinetics in Drosophila mcm5^A7 oocytes. The fertility of mcm5^A7 homozygotes, the absence of the so-called 'spn' phenotype, which is exhibited by mutants that are defective in the repair of DSBs, and the absence of chromosome fragments during prometaphase all argue strongly that these breaks are repaired, but not in a fashion that generates crossover. It is tempting to suggest, as Blanton (2005) has done for mutants in the rec/mcm8 gene, that MCM proteins are required for the processive DNA synthesis that is necessary for the formation of a recombination intermediate, and thus their absence prevents the maturation of a DSB into a mature crossover. Such speculation is bolstered by the observations that while conversion events were at least as frequent in mei-218 and rec/mcm8 oocytes as they are in wild type, the conversion tracts themselves were shorter than observed in wild type. Thus, as suggested by Blanton (2005), perhaps the ability of the oocyte to extend a 3' end in the process of initiating recombination is not sufficient to stabilize a recombination intermediate, but rather 'falls back' to creating a conversion event by a synthesis-dependent strand annealing (SDSA)-like mechanism. This would be plausible if the mcm5^A7 mutation, which is in the C-terminal conserved domain, is required for an interaction with machinery involved specifically in processive DNA replication in meiosis. There are, however, at least two problems with this explanation. First, a further analysis of the mei-218 conversion events failed to confirm an alteration in tract length, and second, at least for mcm5, the ability of mcm5 null alleles to still properly perform endo-reduplicative DNA synthesis, producing normally appearing polytene salivary gland chromosomes, argues against a requirement for at least this protein in general processive DNA synthesis (Lake, 2007).

So then how might these mutants suppress crossing over by a mechanism that is unrelated to their traditional roles in replication? One possibility is that at least Mcm5 is known to regulate transcription via a physical interaction with Stat1 (Zhang, 1998; Snyder, 2005). Thus it is at least possible, even though the mcm5^A7 mutation lies well outside the putative Stat1 interacting domain of Mcm5 (DaFonseca, 2001), that the change created by this mutation impairs the interaction of Mcm5 with Stat1 or some other transcriptional regulator, and in doing so prevents the expression of one or more genes that function in the maturation of DSBs to crossovers (Lake, 2007).

However, on the basis of a recent finding by Shimada (2007) that origin recognition complex proteins in yeast function in the process of establishing and maintaining sister chromatid cohesion, in a fashion that is independent of their role in replication initiation, it is proposed that MCM proteins in flies might play a similar role in meiosis. It is imagined that like the fly Ord and C(2)M proteins, which are thought to be involved in conversion of the cohesion complex into the lateral elements of the synaptonemal complex (for review of this process see Page, 2004), the Mcm5, Rec, and Mei-218 proteins also play a role in the function of axial and/or lateral elements and that it is this defect, rather than a problem in replication per se, that underlies their meiotic defects (Lake, 2007).

It should be noted that this adaptation of the Mcm5 protein for a meiotic function may not be universal. The corresponding mutation to that found in mcm5^A7 has been created in S. pombe and no defect was observed in meiotic recombination (S. Forsburg, personal communication to Lake, 2007). This may reflect the rather unusual constellation of repair and recombination proteins found in Drosophila. Notably lacking from the fly genome are obvious homologs of the Dmc1 protein, which at least in other organisms is required to promote interhomolog exchange events and suppress sister chromatid exchange events. Although it has been have proposed that the missing Dmc1 function might be provided by fly Rad51 homologs, Spn-B and Spn-D, one could imagine that the MCM proteins also play such a role in flies, and thus measurement of meiotic sister chromatid exchange in these mutants would be of real interest. Alternatively, it has recently been demonstrated that the mechanism of recombination in S. pombe is fundamentally different from the double Holliday junction mechanism that prevails in S. cerevisiae, leaving the possibility open either that flies are more like the budding yeast in their mechanism of recombination (in a fashion that makes Mcm5 nonessential for recombination in S. pombe) or that perhaps there are even more than two variations on a theme with respect to the process of meiotic recombination, such that flies have their own unique set patterns of nucleic acid needlework with which to perform crossing over (Lake, 2007).

Finally, it is worth noting that Shima (2007) has recently identified a hypomorphic viable allele of mcm4 that causes chromosome instability and identified a unique function of this protein in tumor suppression in mice. Thus, it is obvious that core MCM proteins play roles outside of DNA replication and that the identification of separation-of-function mutants is going to be essential in elucidating the multiple roles of MCM proteins (Lake, 2007).

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).

Search PubMed for articles about Drosophila Mcm5

Blanton, H. L., et al. (2005). REC, Drosophila MCM8, drives formation of meiotic crossovers. PLoS Genet. 1: e40. PubMed citation: 16189551

Crevel, G., et al. (2007). Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells. PLoS ONE 2(9): e833. PubMed citation: 17786205

DaFonseca, C. J., Shu, F. and Zhang, J. J. (2001) Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-γ. Proc. Natl. Acad. Sci. 98: 3034-3039. PubMed citation: 11248027

Ekholm-Reed, S., Mendez, J., Tedesco, D., Zetterberg, A., Stillman, B., et al. (2004). Deregulation of cyclin E in human cells interferes with prereplication complex assembly. J. Cell Biol. 165: 789-800. PubMed citation: 15197178

Feger, G., et al. (1995). dpa, a member of the MCM family, is required for mitotic DNA replication but not endoreplication in Drosophila. EMBO J. 14: 5387-98. PubMed citation: 7489728

Forsburg, S. (2004). Eukaryotic MCM proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68: 109-131. PubMed citation: 15007098

Lake, C. M., Teeter, K., Page, S. L., Nielsen, R. and Hawley, R. S. (2007). A genetic analysis of the Drosophila mcm5 gene defines a domain specifically required for meiotic recombination. Genetics 176(4): 2151-63. PubMed citation: 17565942

Maiorano, D., Lutzmann, M. and Méchali, M. (2006). MCM proteins and DNA replication. Curr. Opin. Cell Biol. 18: 130-136. PubMed citation: 16495042

Moyer, S. E., Lewis, P. W., Botchan, M. R. (2006). Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. 103: 10236-10241. PubMed citation: 16798881

Page, S. L., and Hawley, R. S. (2004). The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20: 525-558

Schwed, G., May, N., Pechersky, Y. and Calvi, B. R. (2002) Drosophila minichromosome maintenance 6 is required for chorion gene amplification and genomic replication. Mol. Biol. Cell 13: 607-620. PubMed citation: 11854416

Shima, N., et al. (2007). A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nat. Genet. 39: 93-98. PubMed citation: 17143284

Shimada, K. and Gasser, S. M. (2007). The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae. Cell 128: 85-99. PubMed citation: 17218257

Snyder, M., He, W. and Zhang, J. J. (2005). The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation. Proc. Natl. Acad. Sci. 102: 14539-14544. PubMed citation: 16199513

Zhang, J. J., et al. (1998). Ser727-dependent recruitment of MCM5 by Stat1alpha in IFN-gamma-induced transcriptional activation. EMBO J. 17: 6963-6971. PubMed citation: 9843502

date revised: 2 July 2008

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