InteractiveFly: GeneBrief

Minichromosome maintenance 5: Biological Overview | References

BIOLOGICAL OVERVIEW

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).

Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing

The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. Mcm2-7 loading was reconstituted with purified budding yeast proteins. Using biochemical approaches and electron microscopy, it was shown that single heptamers of Cdt1•Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. This work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication (Remus, 2009).

These results provide the first evidence that ORC and Cdc6 load the Mcm2-7 proteins from single Cdt1•Mcm2-7 heptamers into pre-RCs as head-to-head double hexamers. DNA, probably double stranded, passes through the long, central channel of this double hexamer. And, once loaded, the double hexamer is mobile, capable of passive one-dimensional diffusion or 'sliding' along DNA. These features of the pre-RC have implications for how origins are chosen and how replisomes assemble during initiation (Remus, 2009).

The loading of Mcm2-7 requires ORC, Cdc6, and hydrolysable ATP, consistent with requirements in vivo. The requirement for Cdt1 was not tested because it is an integral component of the Mcm2-7 complex. The interaction of Cdt1 with both Mcm2-7 and Orc6 suggests that it may act as a bridge between ORC and Mcm2-7. However, the results demonstrate that Cdc6 is also essential to recruit Mcm2-7 to origins, indicating that additional interactions are involved in this recruitment (Remus, 2009).

Surprisingly, loading of Mcm2-7 in vitro does not require specific ORC binding sites. The results may contribute to resolving the long-standing issue of how orthologs of ORC can act on specific DNA sequences in yeast, but show little or no sequence preference in metazoans. The results indicate that even yeast ORC has no inherent mechanistic requirement for specific DNA sequences in the loading of Mcm2-7. The sequence specific DNA binding of the budding yeast ORC may be an evolutionary adaptation designed to ensure sufficient origin activity in a genome containing very little intergenic DNA. Sequence specificity appears to be an integral part of the S. cerevisiae core ORC while sequence specificity of Schizosaccharomyces pombe ORC is conferred by an extended AT hook domain on the Orc4 subunit. Recruitment of ORC in metazoans may also involve interactions with additional sequence specific DNA binding proteins like TRF2. Consistent with this idea, recruitment of ORC to a GAL4 DNA binding site array via fusion of ORC subunits or Cdc6 to the GAL4 DNA binding domain is sufficient to create a functional replication origin in human cells (Remus, 2009 and references therein).

The binding of Mcm2-7 around double-stranded DNA has implications for how DNA unwinding is ultimately catalyzed by the Cdc45/Mcm2-7/GINS (CMG) complex. Mcm2-7 may act in unwinding analogously to the eukaryotic viral SF3 initiator/helicases including the SV40 large T antigen (TAg) and the papillomavirus E1 protein. The TAg double hexamer can bind to double-stranded DNA, and this binding can induce the generation of a short (8 bp) stretch of melted DNA specifically within one of the two hexamers. Although TAg and E1 can assemble as double hexamers around double-stranded DNA, current models indicate that they act during unwinding as classical helicases by encircling single-stranded DNA. If Mcm2-7 act analogously to these proteins, then CDK-and DDK-dependent events must promote remodeling of the Mcm2-7 complex to encircle single-stranded DNA during origin melting (Remus, 2009 and references therein).

Alternatively, Mcm2-7 may act during replication as a double-strand DNA translocase. In this model, Cdc45 and/or GINS would play a direct, structural role in strand separation, perhaps acting as a 'plough' or 'pin' into which DNA is pumped by Mcm2-7. This is analogous to the bacterial RuvAB Holliday junction branch migrating enzyme in which two RuvB hexamers pump double-stranded DNA through a tetramer of RuvA, which coordinates the separation and reannealing of strands. This second model is favored because it does not require topological reorganization of Mcm2-7 subunits during initiation and because it provides a potential biochemical function for Cdc45 and/or GINS during replication. The helicase activity of archaeal MCM as well as eukaryotic Mcm2-7 complexes on single-stranded DNA substrates need not reflect their mode of action in vivo: even double-stranded DNA translocases like RuvB can function in standard helicase assays, presumably because they can translocate along one strand of DNA and displace annealed oligonucleotides (Remus, 2009 and references therein).

Cohesin organizes chromatin loops at DNA replication factories

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. This study demonstrates that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication (Guillou, 2010).

The first part of this study describes a physical interaction between cohesin and the MCM complex in human cells that is consistent with a previous report of an interaction between Smc1 and Mcm7. Whether the association of cohesin with chromatin depends on the previous formation of pre-RCs at origins has been a matter of discussion. This study shows that cohesin associates normally with chromatin after the down-regulation of ORC or MCM, arguing that cohesin loading is independent of pre-RC formation in human cells, as it happens in yeast or Drosophila cells. Therefore, the requirement of pre-RCs for cohesin loading that has been reported in Xenopus extracts could be a particularity of this system. Xenopus extracts recapitulate the early embryonic cycles, a quick succession of chromosome duplication and segregation events with no active transcription. In this context, the genomic positions where pre-RCs are assembled may constitute the only 'entry points' for cohesin. In addition, considering the results of this study, the loading of cohesin at pre-RC sites in Xenopus would ensure its physical presence around origins, where it would contribute to the dynamics of DNA replication (Guillou, 2010).

Cohesin can be detected at thousands of sites along the genome. While a complete genome-wide correlation between CBSs and replication origins cannot be established because of the lack of a comprehensive map of the latter, using a bioinformatics approach, an enrichment of cohesin at the origins located within the ENCODE representation of the genome has indeed been identified. When data from the cohesin ChIP-chip assay were compared with the genomic positions of origins mapped within ENCODE by nascent strand analyses in the same cell line, it became clear that origins are preferential sites for cohesin binding. This observation, further validated by cohesin ChIP assays, seems a conserved feature through evolution because it has also been reported in yeast, Drosophila (MacAlpine, 2009), and even Bacillus subtilis, and suggests a role for cohesin in origin activity. Actually, it was found that cohesin down-regulation slows down S-phase progression by a mechanism that is independent of sister chromatid cohesion, regulation of gene expression, and checkpoint responses. Instead, single-molecule analyses revealed that cohesin down-regulation reduced the number of active origins and increased the average interfork distance, without affecting fork speed. These results imply that the presence of cohesin at origins modulates their activity, providing a novel link between the DNA replication and cohesion machineries, which is independent from the reported effect of cohesin acetylation on fork progression (Terret, 2009; Guillou, 2010 and references therein).

The assembly of DNA replication factories in human cells entails the physical association of a cluster of origins and the formation of chromatin loops. This study has shown that cohesin down-regulation leads to a significant increase in the length of DNA loops in which chromatin is organized. This result, combined with the negative impact of cohesin loss on DNA replication, leads to a proposal that cohesin is required for the formation and/or stabilization of loops at replication foci. In this model, cohesin would mediate the long-range intrachromosomal interactions necessary to bring together a cluster of replication origins. Loop formation would occur at late mitosis and during G1, at the time of origin selection and licensing. In the resultant structures, origins would be located at the bases of the loops, where they are more prone to fire (Courbet, 2008). Upon cohesin down-regulation, replication foci would be structured in a different manner, with fewer origins, longer loops, and, therefore, larger replicon units. This alternative arrangement explains the S-phase phenotypes and the fact that cohesin down-regulation reduces the average intensity of each replication factory without reducing the total number of replication foci (Guillou, 2010).

Interestingly, down-regulation of CTCF neither delayed DNA replication nor affected halo size. The latter observation may seem surprising, but it could be explained because the 'DNA halo' technique allows the visualization of chromatin loops anchored to insoluble nuclear structures, such as those in replication factories, rather than DNA loops that are formed transiently to regulate transcription. In any case, it is possible that other proteins cooperate with cohesin to organize loops at replication factories, much as CTCF, the mediator complex, or tissue-specific transcription factors cooperate with cohesin to regulate gene expression in different contexts (Guillou, 2010).

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. This report shows that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and it is suggested that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage (Ilves, 2012).

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. This study established that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, it was shown that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, it was demonstrated that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. This study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation (Samel, 2014).

Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. This study aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5^A7. Mcm5^A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis (Hatkevich, 2019).

Accurate segregation of homologous chromosomes during the first meiotic division is essential to reestablish the diploid genome upon sexual fertilization. To ensure faithful meiosis I chromosomal segregation, homologs must become physically connected in part through crossover formation. To enable homolog crossover events, a series of chromosomal and cellular events occur in early meiotic prophase I (Hatkevich, 2019).

During or just prior to the onset of meiosis, homologous chromosomes pair along their entire lengths. Between paired homologs, synapsis, the formation of the synaptonemal complex (SC), ensues. The SC is a tripartite scaffold built between homologs extending the length of the chromosomes and consists of a central region that is nestled between two lateral elements, which are successors of cohesin-based chromosome axes formed between sister chromatids. Coincident with synapsis, DSBs are formed and repaired using a homologous template via homologous recombination (HR), resulting in crossover formation between homologs (Hatkevich, 2019).

Perhaps the most enigmatic event within early meiosis is the mechanism by which a meiotic chromosome selectively pairs and synapses with its unique homologous partner. Initial homolog pairing is believed to be facilitated through early meiotic chromosome movement and telomere or the centromere clustering. However, how homologous pairing is maintained during synapsis to ensure the SC is formed exclusively between homologs is unknown (Hatkevich, 2019).

The model organism Drosophila melanogaster has been used to uncover meiotic mechanisms for over a century. In Drosophila, prior to meiosis, chromosomes enter the germline unpaired; throughout the pre-meiotic region, homologous chromosomes gradually pair. In the nuclei at the last mitotic division prior to meiotic onset (in the 8-cell cyst), centromere-directed chromosomal movements occur, presumably ensuring complete homologous pairing. Also during pre-meiotic mitotic cycles, several proteins, including the cohesin SMC1, are enriched at the centromere. The onset of meiotic prophase I occurs in the 16-cell cyst. At zygotene, the first cytologically resolved stage of prophase, centromeres are clustered into 1 or 2 groups, and the SC nucleates in patches along chromosome arms. As zygotene proceeds into early pachytene, the SC extends between paired chromosomes, yielding full-length SC exclusively between homologs. How these early meiotic events, particularly SMC1 enrichment at the centromere and centromere clustering, contribute to meiotic homologous pairing and synapsis in Drosophila is largely unknown (Hatkevich, 2019).

This study used the Drosophila early meiotic program and a unique genetic mutant to investigate how homolog pairing is maintained during meiotic synapsis. Meiotic homologs in a previously described Drosophila mutant, Mcm5^A7, initially pair, but are unable to maintain pairing during synapsis, suggesting that initial meiotic pairing must be subsequently stabilized by an unknown mechanism to ensure proper synapsis. Using Mcm5^A7 as a genetic tool to interrogate pairing stabilization mechanism(s), it was found that SMC1 localization at the centromere is compromised, correlating with a severe defect in meiotic centromere clustering and a decrease of crossover formation. However, arm cohesion and SC structure appear unperturbed in these mutants. By overexpressing SMC1, this study shows that the defects in centromere clustering, meiotic homolog pairing, homosynapsis, and crossing over in Mcm5^A7 mutants are caused by a lack of centromeric SMC1 localization at meiotic onset. From these results, a model for proper synapsis is suggested in which initial meiotic pairing must be stabilized by centromere clustering, a meiotic event produced by SMC1-enrichment at the centromere and dynamic chromosome movements (Hatkevich, 2019).

At the beginning of this study, it was hypothesized that the crossover defect in Mcm5^A7 mutants was due to a homolog pairing deficiency. FISH results support this hypothesis and revealed that homolog pairing can be lost during synapsis, resulting in seemingly normal SC between heterologous sequences. Centromere-directed chromosome movements occur in Mcm5^A7 mutants, presumably to promote initial chromosome arm pairing; however, centromere pairing and clustering are perturbed. SMC1 enrichment at the centromere is decreased in Mcm5^A7 mutants, while arm cohesion appears normal. Overexpression of SMC1 rescues centromeric-SMC1 localization and downstream meiotic defects, including centromere clustering, pairing, crossover formation, and segregation. From these data, it is proposed that centromeric SMC1 stabilizes initial homolog pairing through centromere clustering, securing meiotic pairing, ensuring homosynapsis and promoting crossover formation (Hatkevich, 2019).

Prior to the onset of meiosis, cohesins are loaded onto centromeres, and homologous chromosomes pair, with arm pairing preceding centromere pairing. Initial homolog pairing is achieved in part by centromere-directed movements in the division prior to meiotic onset. A model in which initial homologous chromosomal pairing is stabilized throughout early meiosis by SMC1-dependent centromere clustering (Hatkevich, 2019).

According to this model, the enrichment of SMC1 at the centromere combined with chromosome movements in pre-meiotic stages yield centromere clustering at meiotic onset. While chromosome arms and centromeres enter meiosis paired, heterologous centromere clustering and/or centromere pairing are required to stabilize pairing during SC assembly. As initial euchromatic SC patches extend along the arms of paired homologs, DSBs are formed and subsequently repaired via HR to yield crossovers, which promote accurate disjunction at the end of meiosis (Hatkevich, 2019).

In Mcm5^A7 mutants, coordinated pre-meiotic centromere-directed movements occur, but a sufficient amount of SMC1 is not localized at the centromere to yield centromere clustering. Thus, at meiotic onset, arms are paired, but centromeres are not clustered. As euchromatic SC nucleation occurs, the stabilization provided by centromere clustering is absent, and homologous loci become unpaired. As synapsis extends, the SC is able to form between nearby chromosomes, regardless of homology, yielding heterosynapsis (intrachromosomal and/or interchromomosomal). DSBs made within regions of heterosynapsis are not repaired via HR due to the absence of an available homologous template. Therefore, crossovers are reduced, and nondisjunction occurs at high frequency in Mcm5^A7 mutants (Hatkevich, 2019).

The SMC1-dependent centromere clustering pairing model highlights the finding that initial meiotic pairing is not sufficient to yield complete homosynapsis. Rather, centromeric SMC1-dependent stabilization must occur after pairing and during synapsis. The inherent requirement of pairing stabilization for proper synapsis suggests that there is a force that opposes homolog alignment during synapsis. Perhaps the SC assembly process itself creates an opposing force that can push paired homologs away from one another in the absence of stabilization; a similar hypothesis was previously proposed in C. elegans. An alternative hypothesis is that recombination, which coincides temporally with synapsis assembly, creates a destabilizing force. However, when meiotic DSBs are eliminated in Mcm5^A7 mutants (as shown through mei-P22 Mcm5^A7 double mutants), homologs are unpaired at a frequency similar to Mcm5^A7 mutants, indicating that the opposing force is independent of recombination. Regardless of the origin of the force, it is proposed that SMC1-dependent centromere clusters act as anchors at the nuclear envelope to maintain the proximity of homologous axes (Hatkevich, 2019).

Although meiotic pairing programs vary among organisms, the SMC1-dependent centromere clustering pairing model may be broadly applicable. In Drosophila and C. elegans, meiotic pairing is independent of meiotic recombination. In contrast, meiotic pairing in organisms such as yeast, plants, and mice require DSB formation (although recombination-independent alignment is required for pairing in these organisms). In DSB-dependent pairing programs, homologs are considered paired at ~400 nm, where DSB-mediated interhomolog interactions can be visualized as bridges. However, contemporaneous with DSB formation, centromeres are coupled or clustered. It is speculated that these centromere interactions stabilize the DSB-dependent arm pairing to ensure synapsis exclusively between homologs in many sexually-reproducing organisms (Hatkevich, 2019).

This study reveals the interesting phenomenon of stable heterosynapsis in Drosophila. Extensive heterosynapsis has been previously reported in C. elegans and yeast with variable SC integrity. Though SC aberrations in Mcm5^A7 mutants cannot be ruled out, the data reveal no structural defects, supporting the notion that 'normal' synapsis is largely homology-independent in Drosophila, as has been observed in C. elegans (Hatkevich, 2019).

In Drosophila, synapsis along the arms initiates as patches during zygotene. In Mcm5^A7 mutants, synapsis initiation between paired homologs appears normal in zygotene but SC elongation fails to be limited to homologous regions. Thus, initiation of synapsis may require homology, but elongation may not. Because this study examined only specific loci and not whole chromosomes, future studies determining the degree of heterosynapsis in Mcm5^A7 mutants may provide more insight into how synapsis and homology interact in flies (Hatkevich, 2019).

While Mcm5^A7 has proven to be a valuable genetic tool, how this particular mutation affects SMC1 localization at the meiotic centromere is unknown. Mcm5^A7 mutants do not affect centromere clustering and pairing in a manner similar to that of mutants that disrupt centromere integrity, such as cal1 Cenp-C double heterozygotes. However, the results do not exclude a role for MCM5 in centromere function or integrity (Hatkevich, 2019).

The canonical role of MCM5 is to function within the replicative helicase, MCM2-7, unwinding double-stranded DNA ahead of the replication fork during S-phase. Because of its important replication role, Mcm5 is an essential gene in every proliferating cell. Numerous studies have shown that replication is required for cohesion localization and establishment, but examining a direct role for any MCM protein in cohesin deposition is difficult since MCMs are essential for replication, which in turn is required for establishing cohesion (Hatkevich, 2019).

Because MCM5 functions within the MCM2-7 replicative helicase, it is tempting to speculate that the Mcm5^A7 mutation may directly perturb SMC1 localization, either through defects in replication or cohesin deposition. No replication defect in Mcm5^A7 mutants has been detected, in either a mitotic or meiotic context. In the future, when individual pre-meiotic nuclei can be isolated from cysts, higher-resolution replication assays may determine whether replication is subtly disrupted in Mcm5^A7 mutants. At this point, however, it seems more likely that the Mcm5^A7 cleanly separates the replication role of MCM5 from a role in meiotic cohesin deposition (Hatkevich, 2019).

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 ID: 16189551

Courbet, S., et al. (2008). Replication fork movement sets chromatin loop size and origin choice in mammalian cells. Nature 455: 557-560. PubMed ID: 18716622

Crevel, G., et al. (2007). Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells. PLoS ONE 2(9): e833. PubMed ID: 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 ID: 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 ID: 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 ID: 7489728

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

Guillou, E., et al. (2010). Cohesin organizes chromatin loops at DNA replication factories. Genes Dev. 24(24): 2812-22. PubMed ID: 21159821

Hatkevich, T., Boudreau, V., Rubin, T., Maddox, P. S., Huynh, J. R. and Sekelsky, J. (2019). Centromeric SMC1 promotes centromer clustering and stabilizes meiotic homolog pairing. PLoS Genet 15(10): e1008412. PubMed ID: 31609962

Ilves, I., Tamberg, N. and Botchan, M.R. (2012). Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. Proc. Natl. Acad. Sci. 109(33): 13163-70. PubMed ID: 22853956

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 ID: 17565942

MacAlpine, H. K., Gordan, R., Powell, S. K., Hartemink, A. J. and MacAlpine, D. M. (2009). Drosophila ORC localizes to open chromatin and marks sites of cohesin complex loading. Genome Res 20: 201-211. PubMed ID: 19996087

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

Remus, D., Beuron, F., Tolun, G., Griffith, J. D., Morris, E. P. and Diffley, J. F. (2009). Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139(4): 719-30. PubMed ID: 19896182

Samel, S. A., Fernandez-Cid, A., Sun, J., Riera, A., Tognetti, S., Herrera, M. C., Li, H. and Speck, C. (2014). A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA. Genes Dev 28: 1653-1666. PubMed ID: 25085418

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 ID: 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 ID: 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 ID: 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 ID: 16199513

Terret, M. E., Sherwood, R., Rahman, S., Qin, J. and Jallepalli, P. V. (2009). Cohesin acetylation speeds the replication fork. Nature 462: 231-234. PubMed ID: 19907496

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 ID: 9843502

date revised: 15 April 2020

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