disc proliferation abnormal


MCM proteins in Xenopus

The Xenopus Cdc6 protein (Drosophila homolog: CG5971) is essential for the initiation of DNA replication. The Xenopus homolog of Yeast and Drosophila ORC2, Xorc2 cannot bind to chromatin at M phase in Xenopus extracts, suggesting that its ability to bind to the origin DNA is regulated by the cell cycle. This difference may be due to the fact that chromosomes of Xenopus and other vertebrates undergo extensive condensation at mitosis. At the end of mitosis, when the chromatin decondenses and the Cdc2-cyclin B complex (for the fly homolog see Cyclin B) undergoes inactivation, both Xorc2 and Xcdc6 can reassociate with chromatin. Binding of Xcdc6 to chromatin requires Xorc2. Following the association of Xorc2 with Xenopus chromatin, the Xcdc6 and Xmcm3 proteins bind shortly thereafter. Xmcm3 cannot bind to chromatin lacking Xcdc6. At some point, S-phase promoting factor (SPF) triggers the firing of the replication origins. In Xenopus, the Cdck2-cyclinE (for a fly homolog see CyclinE) complex is necessary and perhaps sufficient to fulfill the role of SPF. The critical targets of Cdk2-cyclinE may be Xcdc6 and/or components of the ORC. Phosphorylation of Xcdc6 by Cdk2-cyclinE could lead to both the firing of the origin and the subsequent elimination of Xcdc6 from the origin. In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope (Coleman, 1996 and references).

The Xenopus Origin Recognition Complex interacts with the components of the replication licensing system. RLF-M binds to chromatin early in the cell cycle and licenses DNA for replication in the subsequent S phase. Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M also associates with chromatin early in the cell cycle but dissociates during S phase. The assembly of RLF-M into chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle (Rowles, 1996).

An intact nuclear membrane restricts DNA replication to only one round in each cell cycle, apparently by excluding an essential replication-licensing factor throughout interphase. A family of related yeast replication proteins (MCM2, 3 and 5) resembles licensing factor, entering the nucleus only during mitosis. Immunodepletion of a Xenopus homolog of MCM3 (XMCM3) removes a complex of MCM2, 3 and 5 homologs and inhibits replication of Xenopus, sperm nuclei, and permeable G2 HeLa nuclei. However, G1 HeLa nuclei still replicate efficiently. XMCM3 accumulates in nuclei before replication but anti-XMCM3 staining decreases during replication. These results can explain why replicated nuclei are unable to reinitiate replication in a single cell cycle (Madine, 1995a).

The nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but it does prevent the binding of XMCM3 to chromatin. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication. XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. Replication licensing can therefore be divided into two stages. The first requires the entry of a cytosolic "loading factor" excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor that cannot cross the nuclear envelope (Madine, 1995b).

A Xenopus homolog of S. pombe cdc21 exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Xenopus cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci which form prior to reconstitution of the nuclear membrane. The association of cdc21 with chromatin occurs in membrane-free high speed extracts and is resistant to detergent extraction. The spatial organization of the cdc21 foci resembles that of pre-replication centers though no co-localization with RP-A has been observed. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor (Coué, 1996).

Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Xenopus RLF-M consists of all six members of the MCM/P1 protein family (XMcm2-XMcm7). The six MCM/P1 polypeptides co-elute on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitates from purified RLF-M. Further fractionation separates RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provides RLF-M activity. All six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing (Thommes, 1997).

Before initiation of DNA replication, origin recognition complex (ORC) proteins [cdc6, and minichromosome maintenance (MCM) proteins] bind to chromatin sequentially and form preinitiation complexes. After the formation of these complexes in Xenopus laevis egg extracts, and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA replication fails to initiate. After assembly of MCM proteins into preinitiation complexes, removal of the ORC from DNA does not block the subsequent initiation of replication. Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and cdc6 proteins, but dependent on cdk2 activity. Potential targets for this second cdk2 step include phosphorylation of MCM proteins by cdk2, or the cdk2-dependent activation of other proteins essential for initiation, such as cdc7, or an as yet to be identified helicase (Hua, 1998).

Distinct maternal and zygotic genes encode at least one member of the minichromosome maintenance (MCM) protein family. In Xenopus eggs, the MCM2-MCM7 proteins assemble as multimeric complexes at chromosomal origins of replication. The sequential, cell-cycle-dependent assembly of the origin replication complex (ORC), the CDC6 protein and the MCM complex at origins of replication all ensure that DNA replicates only once per cell cycle. The periodic association of the MCM complex with chromatin may be regulated via phosphorylation by cyclin-dependent kinases (Cdks). The first example of a developmentally regulated mcm gene, zygotic mcm6 (zmcm6) now has been cloned. zmcm6 is expressed only after gastrulation, when the cell cycle is remodeled. The zMCM6 protein assembles into MCM complexes and differs from maternal MCM6 (mMCM6) in having a carboxy-terminal extension and a consensus cyclin-Cdk phosphorylation site. There may also be maternal-zygotic pairs of other MCMs. These data suggest that MCMs are critical for cell-cycle remodeling during early Xenopus development (Sible, 1998).

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) and Xenopus Cdc6 (XCdc6). XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first. Although XORC has been shown to be a distinct activity from RLF-B, the relationship between XCdc6 and RLF-B is currently unclear. Active XCdc6 is loaded onto chromatin in extracts with defective RLF, and both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed (Tada, 1999).

In eukaryotic cells, chromosomal DNA replication begins with the formation of pre-replication complexes at replication origins. Formation and maintenance of pre-replication complexes is dependent upon CDC6, a protein that allows assembly of MCM2-7 proteins, which are putative replicative helicases. The functional assembly of MCM proteins into chromatin corresponds to replication licensing. Removal of these proteins from chromatin in S phase is crucial in origins firing regulation. A protein that is required for the assembly of pre-replication complexes has been identified in a screen for maternally expressed genes in Xenopus. This factor (XCDT1) is a relative of fission yeast cdt1 (Drosophila homolog: double parked), a protein proposed to function in DNA replication, and is the first to be identified in vertebrates. Using Xenopus in vitro systems, it has been shown that XCDT1 is required for chromosomal DNA replication. XCDT1 associates with pre-replicative chromatin in a manner dependent on ORC protein and is removed from chromatin at the time of initiation of DNA synthesis. Immunodepletion and reconstitution experiments show that XCDT1 is required to load MCM2-7 proteins onto pre-replicative chromatin. These findings indicate that XCDT1 is an essential component of the system that regulates origins firing during S phase (Maiorano, 2000).

During late mitosis and early interphase, origins of replication become 'licensed' for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Thus, upon metaphase release, some of the geminin is degraded, but the remaining geminin is altered in some way that prevents it from binding and inhibiting Cdt1/RLF-B. The loss of CDK activity allows ORC and Cdc6 to associate tightly with DNA. With all components of the licensing system active, Mcm2-7 is loaded onto origins. Following nuclear assembly, geminin is imported into a functional nucleus and becomes reactivated. This allows it to bind and inhibit Cdt1/RLF-B, thus preventing the relicensing of replicated origins. Since the initiation of replication at licensed origins depends on nuclear assembly, these results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle (Hodgson, 2002).

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication

Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, biotin-streptavidin-modified plasmids were used to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol α, DNA pol δ, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the “unwindosome,” which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex (Pack, 2006; full text of article).

In conclusion, a method has been developed to study replication elongation complexes in vertebrates. By inducing pausing of replication complexes at a defined site on a plasmid, ChIP can be used to interrogate their biochemical composition. MCM2-7, Cdc45, GINS, Mcm10, DNA pol α, DNA pol δ, and DNA pol epsilon are found to localize to the vertebrate DNA replication fork. In the presence of aphidicolin, DNA polymerases and Mcm10 appear to remain bound to the chromatin but are not significantly enriched at the biotin locus, whereas MCM2-7, Cdc45, and Sld5 are highly enriched at the pause site, indicating that a helicase complex is physically uncoupled from the replisome. These data represent the first direct demonstration that MCM2-7, Cdc45, and GINS proteins are associated with the active helicase complex during DNA replication in higher eukaryotes. Consistent with these results, Cdc45, GINS, and MCM2-7 can be coprecipitated from chromatin in Xenopus egg extracts, and a complex of these factors that exhibits helicase activity has been extensively purified from Drosophila eggs. These data argue that the MCM2-7 holocomplex participates in DNA unwinding, since members of all existing subcomplexes (Mcm4/6/7, Mcm3/5, and Mcm2) are present. Therefore, immunofluorescence data, which show a lack of colocalization of MCM subunits with sites of DNA replication, appear to be misleading. The requirement for Cdc45 and GINS during replication elongation suggests that their presence in the helicase complex reported in this study reflects essential auxiliary roles in DNA unwinding, although the mechanism remains to be elucidated. These observations may explain why the MCM2-7 complex by itself exhibits no helicase activity in vitro and why the MCM4/6/7 complex unwinds DNA with low or moderate processivity. It is proposed to call the molecular machine that unwinds DNA at the replication fork the unwindosome, and it is likely that it will contain factors other than Cdc45, GINS, and MCM2-7 (Pack, 2006).

Mammalian MCM proteins

CDC46/MCM5 encodes a protein that is highly conserved among yeast, plants, and animals. It is found in a complex that exhibits DNA replication licensing activity, which is proposed to regulate the synthesis of DNA once and only once per cell cycle. In yeast, loss of function mutations of CDC46/MCM5 decrease DNA synthesis. Very little is known about the regulation of CDC46/MCM5 in any species. In the mouse embryo, expression of cdc46 is increased in unfused portions of the neural tube when the gene encoding the transcription factor, Pax-3 (Drosophila homolog: Paired), is either nonfunctional or underexpressed. These results are observed both in embryos of diabetic mice, which express significantly reduced levels of Pax-3 mRNA, and in Splotch embryos, which carry loss of function Pax-3 alleles. This indicates that expression of cdc46 is negatively regulated as part of a Pax-3-dependent pathway. Since cdc46 appears to regulate DNA synthesis and cell cycle progression, it is possible that its overexpression is involved in defective embryonic development that is associated with loss of Pax-3 function (Hill, 1998).

Human MCM2 is homologous to the yeast nuclear proteins MCM2, MCM3, CDC21, and CDC46. An open reading frame of 1,629 nucleotides encodes 543 amino acids. Sequence comparison reveals 30-40% amino acid identity with each of these four yeast proteins, all of which are considered to play important roles in DNA replication. Human MCM2 gene is expressed ubiquitously in normal tissues. This gene can be localized to chromosomal bands 7q21.3-->q22.1 by fluorescence in situ hybridization (Nakatsuru, 1995).

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. A human Cdc7 homolog, HsCdc7 has been identified as has a human Dbf4 homolog, HsDbf4. HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity. They are low in G1, increase during G1-S, and remain high during S and G2-M. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo 32P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate. Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells (Jiang, 1999).

The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms: chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. A procedure has been established to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. This procedure has been applied to HeLa cells synchronized in each of the G1, S, and G2/M phases; hMCM heterohexameric complexes could be detected in all three phases. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G2/M phase hyperphosphorylation, hMCM2 and 4 in the complexes have been found to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase is inactivated in temperature-sensitive mutant murine FT210 cells, the G2/M hyperphosphorylation of the murine MCM2 and MCM4 and the release of the MCMs from chromatin in the G2 phase are severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association (Fujita, 1999).

All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain, which is conserved from yeast to mammals. MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins have been purified from HeLa cells by using histone-Sepharose column chromatography. The present study has revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities co-purify with a 600-kDa protein complex that consists of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody results in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggests that it migrates along single-stranded DNA in the 3' to 5' direction, and the DNA helicase activity is detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme (Ishimi, 1997).

Minichromosome maintenance (MCM) proteins play an essential role in eukaryotic DNA replication and bind to chromatin before the initiation of DNA replication. MCM protein complexes consisting of MCM2, -4, -6, and -7 bind strongly to a histone-Sepharose column. This interaction has been analyzed at the molecular level. Among six mouse MCM proteins, only MCM2 binds to histone; amino acid residues 63-153 are responsible for this binding. The region required for nuclear localization of MCM2 was mapped near this histone-binding domain. Far-Western blotting analysis of truncated forms of H3 histone indicate that amino acid residues 26-67 of H3 histone are required for binding to MCM2. Mouse MCM2 can inhibit the DNA helicase activity of the human MCM4, -6, and -7 protein complex. These results suggest that MCM2 plays a different role in the initiation of DNA replication than the other MCM proteins (Ishimi, 1998).

A strong body of evidence indicates that cyclin-dependent protein kinases are required not only for the initiation of DNA replication but also for preventing over-replication in eukaryotic cells. Mcm proteins are one of the components of the replication licensing system that permits only a single round of DNA replication per cell cycle. It has been reported that Mcm proteins are phosphorylated by the cyclin-dependent kinases in vivo, suggesting that these two factors are cooperatively involved in the regulation of DNA replication. A 600-kDa Mcm4,6,7 complex has a DNA helicase activity that is probably necessary for the initiation of DNA replication. The in vitro phosphorylation of the Mcm complexes by cyclin A/Cdk2 has been studied to understand the interplay between Mcm proteins and cyclin-dependent kinases. The cyclin A/Cdk2 mainly phosphorylates the amino-terminal region of Mcm4 in the Mcm4,6,7 complex. The phosphorylation is associated with the inactivation of its DNA helicase activity. These results raise the possibility that the inactivation of Mcm4,6,7 helicase activity by Cdk2 is a part of the system for regulating DNA replication (Ishimi, 2000).

Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. A DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. The mouse Mcm2 and Mcm4,6,7 proteins have been expressed and isolated from insect cells and various mutant Mcm4,6,7 complexes have been characterized in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrate that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicate that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicate that the two activities of DNA helicase and single-stranded DNA binding can be separated (You, 1999).

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. A DNA helicase activity has been shown to be associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex has been visualized by electron microscopy after negative staining with uranyl acetate. The complex contains toroidal forms with a central channel and also contains structures with a slit. Gel-shift analysis indicates that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA is comparable to that of SV40 T antigen, although the Mcm4,6,7 complex requires longer single-stranded DNA for the binding than does SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA have been visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes is erased by Mcm2, which is a potential inhibitor of the Mcm4,6,7 helicase. The DNA helicase activity of Mcm4,6,7 complex is inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity (Sato, 2000).

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period was examined using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The results demonstrate that the occurance of MCM binding and the initiation of DNA replication of nuclear chromatin depends on the decrease of Cdc2 activity to critical levels. Similarly, it was found that Cdc2 inhibitory phosphorylation and cyclin B degradation are turned on and off, respectively, depending on the decrease in Cdc2 activity. However, sensitivity of these processes to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle (Iwabuchi, 2002).

Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. Adenoviral vectors have been used to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6 (Cook, 2002).

The best-characterized substrates of cyclin E/Cdk2 are the retinoblastoma family proteins, Rb, p130, and p107. Phosphorylation of Rb by cyclin D/Cdk4 and cyclin E/Cdk2 dissociates Rb from E2F and allows the induction of E2F target genes. The synergy between low-level cyclin E/Cdk2 expression and Cdc6 is only seen when cyclin E/Cdk2 activity is low enough to induce endogenous cdc6 expression minimally. Thus one function of cyclin E/Cdk2 in the induction of S phase is its well-documented role in transcriptional control of E2F target genes such as cdc6. The role of cyclin E/Cdk2 in Mcm chromatin loading is restricted to its function in E2F-dependent transcriptional control of the cdc6 gene because expression of Cdc6 in the absence of cdk activity (either by ectopic expression or by induction of the endogenous gene by E2F) bypasses the need for cdk activity in Mcm chromatin loading. Cyclin E/Cdk2 activity is not required for prereplication complex formation as long as Cdc6 is produced (Cook, 2002).

Clearly, cyclin E/Cdk2 plays additional roles in replication initiation downstream of Mcm chromatin loading because Cdc6-mediated Mcm chromatin loading is not sufficient for replication without cyclin E/Cdk2, and Cdk2 activity is still required for initiation in X. laevis extracts in which transcriptional control is not important. At least one of those functions is likely to be the loading of the Cdc45 protein onto the newly formed prereplication complex, although the precise mechanism of this aspect of cyclin E/Cdk2 function remains to be elucidated (Cook, 2002).

Chromosomal DNA replication requires the recruitment of the six-subunit minichromosome maintenance (Mcm) complex to chromatin through the action of Cdc6 and Cdt1. Although considerable work has described the functions of Cdc6 and Cdt1 in yeast and biochemical systems, evidence that their mammalian counterparts are subject to distinct regulation suggests the need to further explore the molecular relationships involving Cdc6 and Cdt1. Cdc6 and Cdt1 are shown to be mutually dependent on one another for loading Mcm complexes onto chromatin in mammalian cells. The association of Cdt1 with Mcm2 is regulated by cell growth. Mcm2 prepared from quiescent cells associates very weakly with Cdt1, whereas Mcm2 from serum-stimulated cells associates with Cdt1 much more efficiently. Cdc6, which normally accumulates as cells progress from quiescence into G(1), is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region that is essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and evidence is provided that the mechanism of this inhibition involves blocking the binding of Cdt1 to both Mcm2 and Cdc6. These results identify novel molecular functions for both Cdc6 and geminin in controlling the association of Cdt1 with other components of the replication apparatus and indicate that the association of Cdt1 with the Mcm complex is controlled as cells exit and reenter the cell cycle (Cook, 2004).

Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress

In late mitosis and early G1, Mcm2-7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2-7 loaded is in significant excess over the number of origins normally used. This study shows that in human cells excess chromatin-bound Mcm2-7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins are activated within active replicon clusters if replication fork progression is inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2-7 in human cells by RNA interference (RNAi), the use of dormant origins is suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2-7 replicate at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2-7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. It is proposed that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems (Ge, 2007).

Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2-7 loading

The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors, and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. This study investigated the function of the Saccharomyces cerevisiae Orc6, the only ORC subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits prereplicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 (Drosophila homolog: double parked) and to load the Mcm2-7 helicase onto origin DNA. Two regions of Orc6 bind Cdt1 directly, and the extreme C terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of Mcm2-7 loading (Chen, 2007).

back to disc proliferation abnormal Evolutionary Homologs part 1/2

Non-transcriptional control of DNA replication by c-Myc: MCM2-MCM7 subunits, ORC2, Cdc6 and Cdt1, were present in the affinity-purified Myc complex

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. This study shows that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions (Dominguez-Sola, 2007).

Minichromosome maintenance (MCM) proteins are part of the pre-replicative complex, a multiprotein complex essential for the assembly and activity of DNA replication origins13. Indeed, all MCM2-MCM7 subunits, ORC2, Cdc6 and Cdt1, were present in the affinity-purified Myc complex, consistent with the known interaction of Myc with MCM2 and MCM7. In contrast, proteins involved in DNA replication elongation (MCM10, RPA and PCNA) were absent. The interaction with pre-replicative complex components was also observed with N-Myc. Other proteins forming complexes with Myc, such as TRRAP18, were not found in this Myc and pre-replicative-complex-associated complex, whereas small, non-stoichiometrical amounts of Max (Myc-associated factor X) were detectable (Dominguez-Sola, 2007).

Myc and pre-replicative complex proteins co-sedimented in high molecular mass fractions (approx1.7 MDa) after glycerol density gradient sedimentation and size-exclusion chromatography of Myc-bound protein complexes. Notably, Myc is also present in a distinct set of fractions that contained the majority of Max protein that co-purified with this complex). These fractions also contained MCM5, which might be involved in other transcriptional complexes. Overall, these results identify a novel Myc-associated complex in mammalian cells that contains pre-replicative complex components and thus suggests a direct role of Myc in DNA replication (Dominguez-Sola, 2007).

It has been proposed that Myc promotes G1/S transition and DNA replication through the transcription of factors promoting S-phase entry and/or cell growth. The current results indicate that Myc control of DNA replication is not dependent on its transcriptional activity in both Xenopus extracts and mammalian cells. Nonetheless, transcriptional regulation of critical target genes may also be an important component of the overall role of Myc in regulating DNA replication initiation. Notably, the transactivation domain of Myc is required to control both DNA replication initiation and transcriptional activity, suggesting that Myc may use a common molecular mechanism to facilitate both DNA transactions. This mechanism might involve Myc-dependent chromatin modifications such as histone acetylation, which might also be implicated in the selection of replication origins (Dominguez-Sola, 2007).

The results indicate that Myc deregulation generates DNA damage and may promote genomic instability by inducing DNA replication stress, strengthening previous observations. This notion is also supported by the dependence on Werner RecQ helicase for Myc-driven proliferation (Grandori, 2003), and by the requirement for RecQ helicases during replication stress. These observations can explain the occurrence of genomic alterations, such as gene amplification and illegitimate replication of some loci, that are consistently associated with Myc deregulation during tumorigenesis. However, in contrast with other oncogenes that may cause DNA re-replication when deregulated, overexpression of Myc increases the number of active replication origins in the absence of detectable re-replication (Dominguez-Sola, 2007).

The results also suggest that the p53-dependent G2/M checkpoint and subsequent apoptosis observed in mammalian cells carrying deregulated Myc alleles may be due to DNA damage generated predominantly during S phase. Frequent p53 inactivation in tumours carrying deregulated Myc genes may then reflect selection for tumoural cells with disabled checkpoint responses. Thus, these results suggest that Myc may exert its oncogenic function, at least in part, by promoting origin activity, thereby inducing replication stress and genomic instability (Dominguez-Sola, 2007).

Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation

The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. This study addresses the mechanisms that control the specificity of DDK action. Incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, it was found that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. These findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity (Francis, 2009).

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).

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.