disc proliferation abnormal


Other Drosophila MCM proteins

A second MCM gene has been discovered in Drosophila, the fly homolog of yeast MCM2 gene. DmMCM2 is 55% identical in the central 651-amino acid region to yeast MCM2 and 43% identical overall. DmMCM2 is expressed in a pattern corresponding to that of S-phase cells. Mutation inhibits proliferation of cells in the imaginal discs and central nervous system and causes an apparent prolongation of S phase in the embryonic and larval CNS. As with dpa, endoreplicating cells in the gut and salivary glands are able to grow, suggesting a more stringent requirement for MCM function in diploid rather than endoreplicative cells. DmMCM2 is present in the nucleus throughout interphase, suggesting that it resembles the mammalian protein P1 rather than the yeast proteins (Treisman, 1995).

The structural basis for MCM2-7 helicase activation by GINS and Cdc45

Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2-7 helicase onto double-stranded DNA and its activation by GINS-Cdc45 (see Drosophila CDC45L). To better understand these events, the structures of Mcm2-7 and the CMG complex was determined by using single-particle electron microscopy. Mcm2-7 adopts two conformations--a lock-washer-shaped spiral state and a planar, gapped-ring form--in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, these data help explain how GINS and Cdc45 activate Mcm2-7, indicate that Mcm2-7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation (Costa, 2011).

An archaeal MCM protein

Previous studies have identified an ATP-dependent DNA helicase activity intrinsic to the human minichromosome maintenance complex, composed of MCM subunits 4, 6, and 7. In contrast to the presence of multiple MCM genes (at least six) in eukaryotes, the archaeon Methanobacterium thermoautotrophicum DeltaH (mth) genome contains a single open reading frame coding for an MCM protein. The isolation of the mthMCM protein overexpressed in Escherichia coli is reported in this study. The purified recombinant protein exists in both multimeric and monomeric forms. Both forms of the protein bind to single-stranded DNA, hydrolyze ATP in the presence of DNA, and possess 3'-to-5' ATP-dependent DNA helicase activities. Thus, a single mthMCM protein contains biochemical properties identical to those associated with the eukaryotic MCM4, -6, and -7 complex. These results suggest that the characterization of the mthMCM protein and its multiple forms may contribute to an understanding of the role of MCM helicase activity in eukaryotic chromosomal DNA replication (Kelman, 2000).

The minichromosome maintenance (MCM) proteins are essential for DNA replication in eukaryotes. Thus far, all eukaryotes have been shown to contain six highly related MCMs that apparently function together in DNA replication. Sequencing of the entire genome of the thermophilic archaeon Methanobacterium thermoautotrophicum has allowed the identification of only a single MCM-like gene (ORF Mt1770). This gene is most similar to MCM4 in eukaryotic cells. The M. thermoautotrophicum MCM protein has been expressed and purified. The purified protein forms a complex that has a molecular mass of approximately 850 kDa, consistent with formation of a double hexamer. The protein has an ATP-independent DNA-binding activity, a DNA-stimulated ATPase activity that discriminates between single- and double-stranded DNA, and a strand-displacement (helicase) activity that can unwind up to 500 base pairs. The 3' to 5' helicase activity requires both ATP hydrolysis and a functional nucleotide-binding site. Moreover, the double hexamer form is the active helicase. It is therefore likely that an MCM complex acts as the replicative DNA helicase in eukaryotes and archaea. The simplified replication machinery in archaea may provide a simplified model for assembly of the machinery required for initiation of eukaryotic DNA replication (Chong, 2000).

MCM proteins in yeast

MCM2 and MCM3 of S. cerevisiae are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G1 phase, and disappear at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis; their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle (Yan, 1993).

CDC54 is a gene essential for initiation of DNA replication in S. cerevisiae, and which is known to genetically interact with other regulators of the S-phase, including CDC46. CDC54 protein is structurally related to Cdc46p, Mcm2p and Mcm3p by the presence of a conserved domain of 145 amino acids that is internal to each polypeptide. This conserved domain resembles the DEAD box of RNA helicases and is similar to the conserved domain associated with a group of transcription and replication factors with known or assumed DNA-dependent ATPase activity, suggesting it may be involved in nucleic-acid recognition. Comparison of Cdc54p to related proteins from other species reveals that it closely resembles cdc21p from S. pombe (Whitebread, 1995).

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, prompting the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. In contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is the case for related proteins in mammalian cells. It is suggested that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, the phenotype of a gene deletion was examined. cdc21+ is required for entry into S phase; unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events. Defective initiation therefore may impair the coordination between mitosis and S phase (Maiorano, 1996).

The initiation of DNA synthesis is an important cell cycle event that defines the beginning of S phase. This critical event involves the participation of proteins whose functions are regulated by cyclin dependent protein kinases (Cdks). The Mcm2-7 proteins are a family of six conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes. In Saccharomyces cerevisiae, members of the Mcm2-7 family undergo cell cycle-specific phosphorylation. Phosphorylation of Mcm proteins at the beginning of S phase coincides with the removal of these proteins from chromatin and the onset of DNA synthesis. Cdc7-Dbf4 is a Cdk-like serine/threonine protein kinase that is required for the onset of DNA synthesis. Cdc7, the catalytic subunit (see Drosophila Cyclin-dependent kinase 7), is conserved in yeast and mammals and is maintained at a constant protein level throughout the cell cycle. Its protein kinase activity, however, is activated by the regulatory subunit Dbf4 (see Drosophila Chiffon) only during the G1-to-S-phase transition. Bbf4, like cyclins, is expressed periodically during the cell cycle. Dbf4 associates with the A domain of replication origins through other origin-binding proteins (Lei, 1997).

Intact nuclei from G2-phase mammalian cells will replicate their DNA in Xenopus egg extract if they are preexposed to the protein kinase inhibitor 6-dimethylaminopurine in vivo. This competence to rereplicate is accompanied by alterations in the subcellular distribution of the Mcm family of proteins, which are implicated in replication licensing. All family members reassociate with chromatin in G2 cells and this correlates closely with regeneration of replication competence. Newly bound Mcm proteins are functional for replication because, unlike untreated G2 nuclei, replication of treated G2 nuclei in vitro occurs independent of the Xenopus Mcm protein complex. These observations show that the postreplicative state is actively maintained in G2 cells by a protein kinase(s) that regulates the behavior of Mcm family proteins (Coverley, 1998).

In this study, DBF4, which encodes the regulatory subunit of a Cdk-like protein kinase Cdc7-Dbf4, was identified in a screen for second site suppressors of mcm2-1. The dbf4 suppressor mutation restores competence to initiate DNA synthesis to the mcm2-1 mutant. Cdc7-Dbf4 interacts physically with Mcm2 and phosphorylates Mcm2 as well as Mcm3, Mcm4 and Mcm6 in vitro. Blocking the kinase activity of Cdc7-Dbf4 at the G1-to-S phase transition also blocks the phosphorylation of Mcm2 at this defined point of the cell cycle. Taken together, these data suggest that phosphorylation of Mcm2 (and probably other members of the Mcm2-7 proteins) by Cdc7-Dbf4 at the G1-to-S phase transition is a critical step in the initiation of DNA synthesis at replication origins (Lei, 1997).

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p (Drosophila homolog: CG5971), and the MCM proteins are required for establishing the pre-RC. All six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. Mcm3 binds chromatin in G1 and leaves chromatin as cells enter and progress through S-phase, combining with chromatin again at the end of mitosis. McM2 associates with chromatin at the time when the pre-Rc complex is established in G1. At the onset of S-phase, Mcm2 begins to disappear from the chromatin as the pre-RC converts to the post-Rc state. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication (Liang, 1997).

Cdc7p is a protein kinase that is required for G1/S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. The characterization in S. cerevisiae of a recessive mutation is reported in a member of the MCM family, MCM5/CDC46, that bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, these studies suggest that Cdc7p is required late in G1 because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication (Hardy, 1997).

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. Cdc6p then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologs contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6(K114E) protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis (Weinreich, 1999).

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. The assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae is described. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and either mutations in the Cdc6p Walker A and Walker B motifs, or treatment with ATP-gamma-S, inhibit these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, it is proposed that Cdc6p may be an essential determinant of origin specificity in metazoan species (Mizushima, 2000).

Cdc6p is a key factor for the regulation of initiation of DNA replication, but the biochemical function of this protein has been unclear. Studies in vivo suggest that Cdc6p participates in the recruitment of the MCM protein to the pre-RC. It is suggested that it is in this complex that Cdc6p recruits the MCM proteins. Human Cdc6p was shown to contain an intrinsic ATPase activity. Several biochemical activities of Cdc6p that may contribute to its role in initiation of DNA replication have been uncovered. The first of these demonstrates that ORC and Cdc6p interact directly with each other (Mizushima, 2000).

The second type of Cdc6p biochemical activity modulates the DNA binding activity of ORC by restricting DNA binding to functional origin sequences. The increased DNA binding specificity of ORC appears to be accomplished by Cdc6p increasing the rate of dissociation of ORC from nonorigin DNA sequences and by decreasing the rate of association of ORC to nonorigin DNA. Both ATP and a functional nucleotide-binding motif within Cdc6p are required for Cdc6p blocking nonspecific DNA binding of ORC to DNA, suggesting that Cdc6p ATPase activity may be required for this activity. At present it is not known whether Cdc6p is involved in ORC loading on origin DNA in vivo. In vivo footprinting and CHIP assays suggest that ORC is localized to origins throughout the cell division cycle. But it is not known whether both sister chromatids bind ORC at origins immediately after DNA replication, when Cdc6p is not present (Mizushima, 2000).

It is not known when newly synthesized ORC complexes bind to DNA during the cell division cycle in vivo and, therefore, it is not clear when the cooperativity between ORC and Cdc6p results in increased DNA binding specificity. In S. cerevisiae, all six ORC subunits are present at constant levels throughout the entire cell cycle. However, Cdc6p is an unstable protein that varies in amount during the cell cycle, with the protein made just prior to entry into mitosis or in late G1 phase of the cell cycle just before S phase. In rapidly proliferating cells, Cdc6p first binds to chromatin as cells exit mitosis, but in cells that come out of stationary phase Cdc6p is loaded in late G1, prior to activation of the cyclin-dependent protein kinases. Thus it is possible that ORC and Cdc6p interact and bind to vacant origins late in the cell division cycle. The fact that Cdc6p hinders ORC binding to nonorigin sequences in vitro suggests that one function of Cdc6p is to restrict replication only to functional origins. This activity of Cdc6p would increase the probability that all origins have the potential for forming pre-RCs after anaphase of mitosis (Mizushima, 2000).

Cdc6p remains bound to chromatin, most probably via its interaction with ORC, throughout the G1 phase of the cell cycle. During this time, a window exists that allows the MCM complex to bind to origins and form the pre-RC (Mizushima, 2000).

A third biochemical function intrinsic to Cdc6p is its ability to remodel the ORC in vitro so that certain ORC subunits become hypersensitive to protease digestion. The ORC remodeling activity of Cdc6p also seems to require ATP and a functional nucleotide-binding motif in Cdc6p, suggesting that an ATPase of Cdc6p mediates the ORC remodeling (Mizushima, 2000).

It is possible that the conformational change in ORC is involved in recruitment of the MCM proteins and thus, formation of the pre-RC. In support of this suggestion, genetic analyses suggest that ATP binding and possibly ATP hydrolysis activities of Cdc6p are required for the MCM loading on chromatin. Thus it is possible that the remodeled form of ORC is essential for MCM loading, and the role of Cdc6p in MCM loading is indirect via its interaction with ORC. Alternatively, both proteins might cooperate to recruit the MCM complex (Mizushima, 2000).

The identity of the ORC subunits that are remodeled is of considerable interest. On the basis of DNA cross-linking measurements, Orc2p and Orc6p appear to be located on the B2 element proximal side (relative to the A domain) of ARS1, adjacent to the site of initiation of DNA replication. This region of the origin is most likely the site of initial unwinding of the double helix. Although it is not known where the MCM proteins interact with the origin DNA, it is likely that it is near the B2 element for a number of reasons. (1) The MCM proteins have intrinsic DNA helicase activity. (2) The B2 element region of ARS1 has an intrinsic ability to unwind in supercoiled plasmid DNA. Thus it is possible that the MCM protein complex is loaded on to the origin DNA near the B2 element by interacting with ORC that has been remodeled by the ATPase activity of Cdc6p. (3) Because Orc1p, Orc2p, and Orc6p, but not other subunits of ORC are phosphorylated in vitro by the S phase cyclin-CDK Cdc28/Clb5, phosphorylation of these subunits after START may affect the ORC-Cdc6p-ARS DNA interaction and/or the pre-RC (Mizushima, 2000).

Cdc6p, also a target of the cyclin-CDKs, is released from the ORC-origin complex and degraded after cells commit to a new round to DNA replication after passage through START. Therefore, another possibility is that release of Cdc6p from the pre-RC triggers a conformational change in ORC that allows subsequent events during initiation (Mizushima, 2000).

The biochemical activities of Cdc6p in modulating ORC function suggest a way to alter the frequency of origins along chromosomes that has been observed during development in Drosophila and Xenopus. At early stages of development, where origins are close together and the ORC to DNA ratio is relatively high, Cdc6p may not be as important for determining origin specificity. At later stages of development when origins are less frequent along chromosomes, Cdc6p could be an essential component in origin determination. Initial studies with purified ORC from both Drosophila and mammalian ORC suggests that they have relatively weak DNA intrinsic binding specificity. On the basis of the results with yeast ORC, it is suggested that in other eukaryotic species Cdc6p functions with ORC to determine the location of DNA replication origins in chromosomes (Mizushima, 2000).

MCM proteins are a conserved family of eukaryotic replication factors implicated in the initiation of DNA replication and in the discrimination between replicated and unreplicated chromatin. However, most mcm mutants in yeast arrest the cell cycle after bulk DNA synthesis has occurred. The basis for this late S phase arrest was investigated by analyzing the effects of a temperature-sensitive mutation in fission yeast cdc19(+)[mcm2(+)]. cdc19-P1 cells show a dramatic loss of viability at the restrictive temperature, which is not typical of all S phase mutants. The cdc19-P1 cell cycle arrest requires an intact damage-response checkpoint and is accompanied by increased rates of chromosome loss and mitotic recombination. Chromosomes from cdc19-P1 cells migrate aberrantly in pulsed-field gels, typical of strains arrested with unresolved replication intermediates. The cdc19-P1 mutation reduces the level of the Cdc19 protein at all temperatures. The effects of disruptions of cdc19(+ )[mcm2(+)], cdc21(+ )[mcm4(+)], nda4(+ )[mcm5(+)] and mis5(+ )[mcm6(+)] were investigated; in all cases, the null mutants undergo delayed S phase but are unable to proceed through the cell cycle. Examination of protein levels suggests that this delayed S phase reflects limiting, but not absent, MCM proteins. Thus, reduced dosage of MCM proteins allows replication initiation, but is insufficient for completion of S phase and cell cycle progression (Liang, 1999).

Members of the Cdc7 family of protein kinases are essential for the initiation of DNA replication in all eukaryotes, but their precise biochemical function is unclear. The fission yeast Cdc7 homolog Hsk1 has been purified approximately 30,000-fold, to near homogeneity. Purified Hsk1 has protein kinase activity on several substrates and is capable of autophosphorylation. Point mutations in highly conserved regions of Hsk1 inactivate the kinase in vitro and in vivo. Overproduction of two of the mutant hsk1 alleles blocks initiation of DNA replication and deranges the mitotic checkpoint, a phenotype consistent with a role for Hsk1 in the early stages of initiation. The purified Hsk1 kinase can be separated into two active forms, a Hsk1 monomer and a heterodimer consisting of Hsk1 complexed with a co-purifying polypeptide, Dfp1. Association with Dfp1 stimulates phosphorylation of exogenous substrates but has little effect on autokinase activity. Dfp1 has been identified as the fission yeast homolog of budding yeast Dbf4. Purified Hsk1 phosphorylates the Cdc19 (Mcm2) subunit of the six-member minichromosome maintenance protein complex purified from fission yeast. Since minichromosome maintenance proteins have been implicated in the initiation of DNA replication, the essential function of Hsk1 at the G1/S transition may be mediated by phosphorylation of Cdc19. Furthermore, the phosphorylation of critical substrates by Hsk1 kinase is likely regulated by association with a Dbf4-like co-factor (Brown, 1999a).

In fission yeast, the Hsk1 protein kinase is essential for the initiation of DNA replication. Hsk1 forms a heterodimeric complex with the regulatory subunit, Dfp1. Reconstitution experiments with purified proteins indicate that Dfp1 is necessary and sufficient to activate Hsk1 phosphorylation of exogenous substrates, such as the Schizosaccharomyces pombe minichromosome maintenance protein Cdc19. The dfp1(+) gene is essential for viability of S. pombe, and depletion of the Dfp1 protein significantly delays the onset of S phase. Dfp1 is a phosphoprotein in vivo and becomes hyperphosphorylated when cells are blocked in S phase by treatment with the DNA synthesis inhibitor hydroxyurea. Hyperphosphorylation in S phase depends on the checkpoint kinase Cds1. The abundance of Dfp1 varies during progression through the cell cycle. The protein is absent when cells are arrested in G(1) phase. When cells are released into the cell cycle, Dfp1 appears suddenly at the G(1)/S transition, coincident with the initiation of DNA replication. The absence of Dfp1 before S phase is due largely, but not exclusively, to posttranscriptional regulation. It is proposed that cell cycle-regulated activation of Dfp1 expression at the G(1)/S transition results in activation of the Hsk1 protein kinase, which, in turn, leads to the initiation of DNA replication (Brown, 1999b).

An in situ technique is described for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. This method was applied to the DNA replication factor mcm4/cdc21. Chromatin association is found to occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, it has been shown that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication (Kearsey, 2000).

In both budding and fission yeasts, it is likely that the timing of MCM chromatin association and pre-RC assembly is determined by the kinetics of CDK inactivation during mitosis. In budding yeast, Cdc28 kinase activity inhibits nuclear accumulation and chromatin association of MCMs, together with the assembly of pre-RCs. Cdc28 is associated with B-type (Clb) cyclin partners during mitosis, and B-cyclin degradation starts at the metaphase to anaphase transition, although persistence of Clb2-Cdc28 activity during anaphase is likely to inhibit pre-RC formation until the end of mitosis. In fission yeast, transcription of cdc18+ begins at metaphase, but cdc18 protein cannot accumulate at this point owing to its destabilization by cdc2 phosphorylation. Cdc13, which is the major mitotic B-type cyclin partner of cdc2, is degraded during mid-anaphase, and this is likely to trigger mcm4 chromatin binding at this point by allowing accumulation of cdc18 protein. It remains to be established whether other components of fission yeast pre-RCs associate with chromatin with similar kinetics to mcm4 (Kearsey, 2000).

The data suggest that pre-RC formation in fission yeast occurs earlier during mitosis than in budding yeast and mammalian cells. This is likely to be important, as the G1 phase of the S. pombe cell cycle is very short and the initiation of DNA replication occurs soon after mitotic exit. Mitotic pre-RC formation may also occur in other eukaryotic cell cycles where the G1 phase is very short or non-existent, such as in early Xenopus or Drosophila development, when embryonic cells cycle rapidly between S and M phases. Early in Xenopus development, individual chromosomes become surrounded by a membrane during anaphase, to form karyomeres. MCM proteins accumulate within such karyomeres, and it is therefore possible that chromatin association may also occur during anaphase. In the plasmodial phase of Physarum, the absence of a G1 phase presumably also requires MCM chromatin association to occur in anaphase, to allow DNA replication to commence in telophase. The assembly of pre-RCs during anaphase implies that chromosome condensation during this phase of the cell cycle does not bar access of MCM and Cdc18 proteins to chromatin. In this regard, it is possible that mitotic chromosome organization may have an influence on replication origin distribution, and it will be interesting to determine whether mitotic pre-RC assembly requires additional factors not necessary for pre-RC formation in G1 (Kearsey, 2000 and references therein).

Many proteins, of which pre-RC components are just one class, show periodicity in chromatin binding: the assay described here should be generally useful for their analysis. These include other replication components such as DNA polymerase, and cohesins, which play a key role in the regulation of chromatin segregation during mitosis (Kearsey, 2000 and references therein).

In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. In living cells, the Mcm proteins are found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 are concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal makes all Mcm proteins constitutively nuclear. In the absence of functional Cdc6 (Drosophila homolog: CG5971), Clb/Cdc28 kinases are necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincides with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases are required for Mcm-GFP export. It is proposed that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. It is further proposed that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus (Nguyen, 2000).

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18, which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and a re-replication assay has been used to identify Cdt1 (Drosophila homolog: double parked) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants, suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes (Nishitani, 2000).

The six MCM (minichromosome maintenance) proteins are essential DNA replication factors: each contains a putative ATP binding motif and together form a heterohexameric complex. These motifs are required for viability in vivo and the coordinated hydrolysis of ATP in vitro. Mutational analysis discriminates between two functionally distinct MCM protein subgroups: Mcm4p, 6p, and 7p contribute canonical ATP binding motifs essential for catalysis, whereas the related motifs in Mcm2p, 3p, and 5p serve a regulatory function. Reconstitution experiments indicate that specific functional interactions between these two subgroups are required for robust ATP hydrolysis. These observations show parallels between the MCM complex and the F1-ATPase; also discussed is how ATP hydrolysis by the MCM complex might be coupled to DNA strand separation (Schwacha, 2001).

There are two possible explanations for the synergistic stimulation in ATPase activity afforded by interaction between the two MCM subgroups: (1) the ATP hydrolysis sites might comprise residues from specific subunits of each subgroup with Mcm4/6/7p contributing the Walker A box residues (a glycine-rich sequence containing an invariant lysine that functions to bind the phosphates of the nucleoside triphosphate) and Mcm2/3/5p contributing additional unknown residues; (2) Mcm2/3/5p might act as a positive activator of Mcm4/6/7p. Both possibilities have precedence but currently it is not possible to distinguish between them. Shared active sites are observed in other ATPases, and proteins that positively regulate the activity of small regulatory GTPases are common (Schwacha, 2001).

It is proposed that three active sites for ATP hydrolysis are formed at interfaces between Mcm2/3/5p and Mcm4/6/7p. Coincubation experiments suggest that Mcm3p with Mcm7p, and Mcm2p with Mcm6p, interact to form an active ATPase site. It is speculated that Mcm4p and Mcm5p interact to form a third functional dimer. Although such a complex has not yet been isolated, allele-specific genetic interactions between Mcm4p and Mcm5p have been demonstrated (Schwacha, 2001).

To reconcile the available biochemical and genetic data, it is proposed that the MCM complex is a circular molecule composed of stacked trimers. The results imply functional interactions between Mcm3p and Mcm7p, and Mcm2p and Mcm6. In contrast, physical interactions among the MCM subunits have been demonstrated by isolated subcomplexes containing Mcm4/6/7p ± Mcm2p and the dimer Mcm3/5p. Electron microscopy provides further structural constraints, indicating that the MCM subunits form a hexameric ring . These contrasting observations can be unified if the MCM complex is composed of a trimer of Mcm2/3/5p stacked on a trimer of Mcm4/6/7p. Rotation between the trimers of 60° will form a ring in which each subunit contacts four other subunits. This structure facilitates both the functional and physical interactions implied in the current and previous studies and is consistent with prior models of MCM structure. Such a structure might be expected to appear abnormally large by gel filtration, consistent with the analysis of the MCM complex as well as a similar analysis of the Xenopus MCM complex. The interdigitation of three catalytic sites between three noncatalytic sites is also a feature of the F1-ATPase (Schwacha, 2001).

The MCM hexamer shares many unexpected parallels to the highly studied F1-ATPase. In addition to previously discussed features, several kinetic features of the MCM complex can be rationalized by comparison to the F1-ATPase. It is proposed that single mutant complexes cause a blockage to ordered ATP hydrolysis, and the complex ATP hydrolysis kinetics represent sequential occupancy of the active sites. Kinetic parallels between the F1-ATPase and the T7 helicase have also been reported (Schwacha, 2001).

By analogy to the F1-ATPase, ordered ATP hydrolysis may normally occur within the MCM hexamer. In contrast to the MCM complex, the F1-ATPase is composed of only two different subunits (alpha and beta), making mutational analysis difficult. However, chemical modification and reconstitution experiments demonstrate that loss of a single active site in the F1-ATPase blocks the ATP hydrolysis of the entire complex. These results reflect an ordered ATP hydrolysis cycle such that one mutant subunit can block the firing order of the entire complex. This explanation is consistent with the results with single-mutant MCM hexamers. It is further proposed that the residual ATPase activity in the single-mutant hexamers represents 'stuttering' of a single subunit or pair of subunits at the point of blockage in the hydrolysis cycle. Since additional mutations in Mcm2/3/5p suppress this effect, Mcm2/3/5p may normally function to coordinate a strict hydrolysis order among the active sites. It is suggested that multiple mutations in the Walker A motif of Mcm2/3/5p uncouple ATP hydrolysis from the wild-type hydrolysis order (Schwacha, 2001).

The MCM complex may allosterically function similarly to the F1-ATPase. There are two striking features of the ATP hydrolysis kinetics of the MCM complex: (1) the higher the concentration of ATP, the harder it is to fill the remaining sites in the complex and (2) ATP hydrolysis is disproportionately stimulated by high concentrations of ATP. Both features are observed in the F1-ATPase. As has been shown for the MCM complex, the F1-ATPase has high, medium, and low affinity ATP hydrolysis modes, with the medium affinity mode likely to represent the physiologically active state of the enzyme. The analogous affinity mode in the MCM protein has a Km for ATP of ~1 mM, well within physiological ATP concentrations of ~3 mM. The binding change model explains these multiple modes of ATP hydrolysis by the sequential binding, hydrolysis, and release of ATP from the three active sites in the ß subunits. The high-affinity/low-activity state represents hydrolysis of a complex with only one bound ATP. The binding of an additional ATP molecule stimulates the turnover rate of the first bound ATP, resulting in the medium-affinity/medium-activity mode. The filling of all three sites with ATP further stimulates hydrolysis and results in the low-affinity/high-activity mode. With the MCM proteins, this effect apparently does not require the Walker A motifs on Mcm2/3/5p, since the Mcm2/3/5K->Ap triple-mutant complex has kinetic properties similar to the wild-type. This suggests that the Mcm4, 6p, and 7p subunits are not only in physical contact, but can also directly regulate one another independent of the Walker A motifs on Mcm2p, 3p, and 5p (Schwacha, 2001).

The F1-ATPase contains a central cavity that encloses the gamma protein. Sequential binding and hydrolysis of ATP causes rotation of the gamma subunit. By analogy, the MCM complex may bind DNA within its central channel and couple ATP hydrolysis to rotational unwinding of the DNA. However, neither DNA helicase activity nor DNA stimulation of the MCM ATPase activity with either the intact MCM hexamer or the Mcm4/6/7p subassembly from S. cerevisiae has been observed. Although helicase activity has not been observed for the intact hexamer from any system, a poorly processive helicase activity unique to the Mcm4/6/7p subassembly has been reported. These results suggest that Mcm4/6/7p is essential for helicase activity, whereas Mcm2/3/5p serves to coordinate and perhaps negatively regulate this activity. However, the ability of Mcm2/3/5p to stimulate the ATPase activity of Mcm4/6/7p suggests that the entire heterohexameric complex functions coordinately. The absence of in vitro helicase activity from the intact heterohexamer may be explained by the lack of a required loading factor, as has been suggested for S. pombe and Xenopus, or specific posttranslational modifications absent in the expression system used in this study (Schwacha, 2001).

Analysis of ATP hydrolysis by the MCM complex has allowed a dissection of MCM function and has provided a plausible explanation for the contrasting observations concerning this complex. These data strongly support the hypothesis that Mcm4/6/7p is the 'catalytic core' of the MCM complex, but clearly indicate that the Mcm2/3/5p subgroup has essential roles in both the positive and negative regulation of Mcm4/6/7p. These results strongly support the idea that the MCM heterohexamer is the active in vivo species and underscore an essential role for ATP hydrolysis by the MCM complex during DNA replication (Schwacha, 2001).

Cdt1 is essential for loading Mcm2-7 proteins into prereplicative complexes (pre-RCs) during replication licensing and has been found in organisms as diverse as fission yeast and humans. A homologue of Cdt1 has been identified in Saccharomyces cerevisiae, that is required for pre-RC assembly. Like Mcm2-7p, Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by cyclin dependent kinases (cdks). Cdt1p interacts with the Mcm2--7p complex, and the nuclear accumulation of these proteins during G1 is interdependent. This coregulation of Cdt1p and Mcm2-7p represents a novel level of pre-RC control (Tanaka, 2002).

CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6

In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. Helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an 'RXL' cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition (Chen, 2011).

MCM proteins in Xenopus

Continued: see disc proliferation abnormal Evolutionary Homologs part 2/2

disc proliferation abnormal: Biological Overview | Regulation | Developmental Biology | References

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