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Proc Natl Acad Sci U S A. Jul 5, 2006; 103(27): 10236–10241.
Published online Jun 23, 2006. doi:  10.1073/pnas.0602400103
PMCID: PMC1482467

Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase


The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2–7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2–7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.

Keywords: Drosophila, ATPase

Although in recent years there has been significant progress in our understanding of the molecular mechanisms of eukaryotic DNA replication, the identity of the primary activity that unravels duplex DNA at the growing fork has remained unclear. Based on a range of indirect data, there is widespread support for the idea that the Mcm2–7 hexamer constitutes part of the replicative helicase (1). Each of the genes is essential, and five of six Mcm2–7 proteins have been shown to be required for both DNA replication initiation and elongation in vivo in Saccharomyces cerevisiae (2, 3). Assays with Xenopus cell-free extracts have shown that all Mcm2–7 proteins are required for chromosomal DNA unwinding and that Mcm2, Mcm5, and Mcm7 localize to sites of DNA unwinding on a plasmid (46). The primary structure of the proteins places each in the helicase supergroup of the AAA+ family, and archaeal forms do show robust helicase function (79). Curiously, the purified Mcm2–7 complex has not displayed helicase activity in vitro; in contrast, the subcomplex of Mcm4, Mcm6, and Mcm7 does (1012). Moreover, the activity of this subcomplex is inhibited by Mcm2, Mcm3, or Mcm5 (10, 11, 13, 14). Intricate models involving continuous assembly and rearrangements of Mcm proteins at the replication fork could somehow accommodate these findings. A simpler hypothesis posits that other proteins are required to activate the Mcm 2–7 complex for helicase function.

The protein Cdc45 has emerged as a pivotal factor in the G1 to S phase transition and as a possible helicase cofactor (4, 6, 15, 16). CDC45 is an essential gene product in yeast (17) and engages proteins assembled in the prereplication complex at origin sites on the DNA during S phase as origins are activated (16, 18). Further, in Xenopus egg extracts, Cdc45 associates with sites of DNA unwinding on a plasmid (6), and interfering antibodies directed at Cdc45 abolish chromosomal unwinding (4). There is a conserved genetic interaction between Cdc45 and the Mcms; specific mutations in these genes can either suppress or show synthetic lethality with the other (1921). By coimmunoprecipitation/immunoblot experiments, Cdc45 has been shown to associate with a variety of other DNA replication proteins, including DNA polymerase α (22, 23), various Mcm2–7 proteins (16, 23, 24), Mcm10 (25), Orc2 (26), Sld3 (27), and Sld5 (28). Many of these interactions may be important but transient or indirect and may obscure a core function or complex within which Cdc45 works.


Purification and Identification of the Cdc45/Mcm2–7/GINS (CMG) Complex.

A protocol to purify a high-molecular-weight complex containing the Cdc45 protein from Drosophila embryo extracts is outlined in Fig. 1A (see Materials and Methods and see also Table 1 and Supporting Text, which are published as supporting information on the PNAS web site, for additional details). Final purification used immunoprecipitation (IP) of Cdc45 protein from the peak Cdc45-containing fractions of the Mono Q column and elution from the antibody resin with buffer (pH 2.5). Before elution, the material in the IP was extensively washed with buffer containing 1 M KCl and treated with ethidium bromide and MNase to remove any contaminating DNA. As shown in Fig. 1B and Fig. 6, which is published as supporting information on the PNAS web site, Cdc45 coimmunoprecipitates with 10 other major proteins. The identity of the copurifying proteins was determined by two methods: (i) mass spectroscopy of individual protein bands cut out from the gel (see List 1, which is published as supporting information on the PNAS web site) and (ii) immunoblotting of the eluate with protein-specific antibodies.

Fig. 1.
Purification of the CMG complex. (A) Purification schematic. See the supporting information for additional details. (B) Material eluted from the anti-Cdc45 IP after the Mono Q step was separated by SDS/PAGE and visualized by Deep Purple stain. For improved ...

The 10 binding partners of Cdc45 were found to be the six proteins of the Mcm2–7 complex and CG14549, CG9187, CG18013, and CG2222, which blast searches revealed are the Drosophila homologs of the four members of the GINS complex (Sld5, Psf1, Psf2, and Psf3, respectively) (Fig. 1B). We also performed mass spectroscopy analysis on the total protein pool obtained after an anti-Cdc45 IP from the Cdc45-containing DEAE fractions from the protocol described in Fig. 1A (see Table 2, which is published as supporting information on the PNAS web site). The only DNA replication proteins identified by this method were also Cdc45, Mcm2–7, and GINS.

Sld5, the founding member of the GINS complex, was first uncovered in a S. cerevisiae synthetic lethal screen with Dpb11 (29). Dpb11 was itself genetically isolated in a suppressor screen for a mutant in a subunit of DNA polymerase ε (30). The Psf genes are each partners of Sld5 and are required for DNA synthesis in yeast and in Xenopus cell-free DNA replication extracts (28, 29). The GINS proteins form a tetramer, and it was recently shown that Sld5 localizes to sites of DNA unwinding on a plasmid in Xenopus egg extracts (6), suggesting that all four GINS proteins may be present in a DNA helicase complex.

Examination of the Superdex-200 column fractions from Fig. 1A shows that the majority of the Cdc45 protein in these extracts exists as a free, low-molecular-weight pool (see Fig. 1C and Fig. 7, which is published as supporting information on the PNAS web site). However, only discrete high-molecular-weight fractions contain a complex that shows an interaction among Cdc45, the GINS members, and the Mcm proteins. Fig. 1C shows an immunoblot of the material eluted from an anti-Cdc45 IP from each Superdex-200 chromatography fraction. The size of the complex as estimated from the elution position on the column closely matches the sum of the individual components (see Discussion). We were not able to reform the high-molecular-weight complex by mixing of lower molecular-weight fractions, which contain an excess of the components (see Fig. 7). We estimate that ≈5% of the total Cdc45 and GINS proteins and ≈1% of the total Mcm proteins in our extracts are in this complex. These results suggest a role for specific protein modifications that would allow for such associations and would support a previous report that the Cdc45 protein does not interact with individual Mcm2–7 proteins (31).

To further explore the notion that a discrete 11-member CMG complex exists, we embarked on independent purification methods. We prepared 0- to 12-h embryo extracts from Drosophila expressing a functional, FLAG-tagged version of Mcm6 (32). A single-step affinity purification of FLAG-Mcm6 from these flies captures mainly FLAG-Mcm6 in complex with Mcm2, Mcm4, and Mcm7 (Fig. 2A Left). This result was anticipated because subcomplexes of the Mcm proteins are in vast (>100-fold) excess to the high-molecular-weight complex we had uncovered. IP of Cdc45 from the purified FLAG-Mcm6 material yielded the complete CMG complex (Fig. 2A Right). In another set of experiments, we fractionated embryo extract, following the high-molecular-weight Cdc45 through the Mono Q step as in Fig. 1A, and with antibodies directed against Mcm5, Sld5, Psf2, and Psf3, asked whether the individual reagents would in turn precipitate the proteins identified by previous methods. Fig. 2B shows a Deep Purple stain of the material eluted from the anti-Psf2 IP. Cdc45 and the complete Mcm2–7 complex coimmunoprecipitated with the GINS complex, and immunoblots of the material eluted from each IP (see Fig. 8, which is published as supporting information on the PNAS web site) confirmed the tight association of each of the 11 members of this complex. Based on these multiple lines of evidence, we conclude that Cdc45, Mcm2–7, and GINS form a stable, high-molecular-weight biochemical unit, which we refer to as the CMG complex. An immediate question was whether the complex contained an active form of the hypothetical Mcm2–7 helicase.

Fig. 2.
IPs with antibodies against different CMG complex members. In this figure, the proteins were identified by immunoblot and Rf value. (A) (Left) FLAG-Mcm6 material purified from embryo extracts by anti-FLAG chromatography. (Right) From the material first ...

Purification of the CMG Complex by Peptide Elution from an Affinity Resin.

We attempted to purify the CMG complex to homogeneity by conventional chromatography but found that the only biochemical step that accomplished this purification was an anti-Cdc45 affinity resin. Thus, to assay the CMG complex for helicase activity, we sought to release the CMG complex from antibodies with a gentle elution method that did not abolish any intrinsic enzymatic function. We searched for peptide-specific anti-Cdc45 antibodies and explored releasing the intact CMG complex from the anti-Cdc45 antibody beads by peptide elution (see Fig. 9A, which is published as supporting information on the PNAS web site). We selected two peptides from a region of Cdc45 that is hydrophilic and predicted to have no secondary structure (see Fig. 9B). Purified antibodies specific to peptide B can immunoprecipitate Cdc45 protein and release Cdc45 when challenged with excess free peptide B in a neutral buffer (see Fig. 9C). Importantly, the anti-Cdc45 peptide B antibodies can also bind and release Cdc45 protein that is in the CMG complex (Fig. 3C), indicating that this region is on the surface of the Cdc45 protein and that it is exposed on both free Cdc45 protein and Cdc45 protein in the CMG complex.

Fig. 3.
The CMG complex is a helicase. (A) Schematic of the purification protocol used for the helicase assay. (B) Illustration of helicase substrate (not to scale). (C) (Top) PhosphorImager picture of helicase assays with anti-Cdc45 peptide B-purified material ...

Identification of CMG-Associated Helicase Activity.

We used the anti-Cdc45 peptide B-specific antibodies to purify the CMG complex according to the procedure outlined in Fig. 3A and asked whether the purified material had helicase activity. The substrate used contains a 40-bp duplex region with a short tail annealed to a single-stranded circle (Fig. 3B). Fig. 3C shows that helicase activity peaks with the protein peak of the purified CMG complex. Fig. 3C Left shows a Deep Purple stain of the material purified by this protocol from Mono Q fraction 20, indicating the presence of the complete CMG complex.

Immunodepletion of column fractions with specific antisera raised to recombinant proteins showed that the activity we had assayed above was directly associated with the CMG complex. We pooled material from Mono Q fractions 18–21, divided it into three portions, and then immunodepleted it with specific antibodies. The antibody beads were all prepared in the same buffer to minimize the possibility of a nonspecific inhibitor being present with any of the antibody resins. Fig. 3D Left examines the extent of depletion in the material treated with control, anti-Psf2, or anti-Mcm5 antibodies. We were able to immunodeplete Psf2 protein to levels below detection and to at least 50% of the starting level for Mcm5 protein. From the immunodepleted material, we attempted to purify the CMG complex with anti-Cdc45 peptide B beads, and the respective eluted proteins were tested for helicase activity. Fig. 3D Right shows that the Psf2-depleted material contains no Cdc45-associated helicase activity, and the Mcm5-depleted material has reduced Cdc45-associated helicase activity, consistent with the reduction, but not complete removal, of Mcm5 protein from the starting protein pool. The material depleted with control antisera showed robust Cdc45-associated activity. These results demonstrate that the helicase activity observed in Fig. 3C is associated with the CMG complex and that Psf2 and Mcm5 are components of the active helicase complex.

Helicase activity was also identified in CMG complex isolated from purified FLAG-Mcm6 material (Fig. 3E Left and Center). Anti-Cdc45 peptide B resin was mixed with purified FLAG-Mcm6-containing complexes, the resin was washed extensively, and bound CMG complexes were eluted with a neutral buffer containing Cdc45 peptide B. To ensure that any observed helicase activity in these preparations was not from residual Mcm4,6,7 complex that may have not been completely washed from the resin after binding CMG to the anti-Cdc45 peptide B beads, we mock-eluted with buffer containing FLAG peptide before eluting with buffer containing Cdc45 peptide B. Fig. 3E Left shows helicase assays with the material eluted from the anti-Cdc45 peptide B resin by the mock elution or the Cdc45 peptide B elution and indicates that the detected helicase in these assays is associated with the CMG complex. We have found in our various preparations of the CMG complex that the helicase activity is ATP-dependent (Fig. 3E Center) and saturates with 1 mM nucleotide (Fig. 3E Right).

Two substrates (Fig. 4A Left) were used to test the directionality of the CMG complex tracking movement on DNA. As indicated by the displacement of the 5′ labeled substrate in Fig. 4A Right, the CMG complex translocates on DNA in a 3′–5′ direction. We have also determined that the CMG complex will displace a substrate identical to that in Fig. 3B, except with a 3′ 30T tail instead of a 5′ tail (data not shown). Thus, the CMG complex can load onto helicase substrates with a gapped single strand (Fig. 4A) or 5′ or 3′ overhangs, like Mcm4,6,7 (10, 33).

Fig. 4.
CMG directionality and processivity. (A) (Left) Cartoon of procedure for preparing substrates for directionality assays. A 48-mer primer was annealed to m13mp18 ssDNA plasmid and then labeled with 32P at the 5′ end with T4 polynucleotide kinase ...

To assay the processivity of the CMG helicase, we annealed the primer used in Fig. 3B to m13mp18 ssDNA plasmid and extended the primer with T7 DNA polymerase to generate a population of duplex primer–plasmid regions of various and heterogeneous lengths. Fig. 4B shows the results of these helicase assays with titrations of the CMG complex, and we conclude that the complex can work processively for many hundreds of base pairs. From analysis of side-by-side comparisons with the Drosophila Mcm4,6,7 subcomplex, we conclude that the CMG complex is as processive as the subcomplex and perhaps more active at lower levels of protein.

In Vivo Requirement of Cdc45 and GINS.

The four proteins of the GINS complex are required for cell cycle progression in yeast (29), but parallel in vivo or cell-based data have not been reported for most GINS members in metazoans. We depleted Cdc45 or the GINS complex members from Drosophila Kc tissue culture cells by RNA interference (RNAi) and examined the cell cycle distribution of the treated cells. Loss of Cdc45 or any one of the GINS complex members results in a significant impairment of cell cycle progression and an accumulation of cells in G1 and S phase (Fig. 5A). These results support a recent report that Psf1 is required for embryo development and cell proliferation in mice (34) and indicate an in vivo requirement for GINS during S phase in metazoans.

Fig. 5.
Cdc45 and GINS proteins are required for normal S phase progression. (A) (Left) Drosophila Kc tissue culture cells were treated with ≈500 bp of dsRNA directed against Cdc45, Sld5, Psf1, Psf2, Psf3, or nonspecific (NS) RNA. After 8 days of RNAi ...


We have provided evidence that Cdc45 exists in a stable biochemical unit with the Mcm2–7 and GINS complexes and that this large complex has associated with it an ATP-dependent helicase activity. The evidence that the Mcm2–7 complex is responsible for this activity (as opposed to another helicase or a subset of the Mcm proteins) is not definitive, but it is an attractive hypothesis. Reconstitution of the complex from recombinant proteins will be the next step in testing this notion. The identification of the CMG complex and its associated helicase activity supports previous reports that implicate the Mcms, Cdc45, and GINS in chromosome unwinding (46), and it begins to provide a molecular model for the mechanism of DNA unwinding at the eukaryotic DNA replication fork.

Formation of the CMG Complex.

Cdc45 and GINS first associate with replication origins at the G1 to S phase transition after the activation of the cyclin-dependent kinase (Cdk) and Cdc7 protein kinases (16, 35), and it is possible that phosphorylation of one or more of the CMG complex members by Cdk and/or Cdc7 may promote Cdc45 and GINS association with Mcm2–7. Mcm proteins have been shown to be phosphorylated at the G1 to S phase transition (36, 37), and Cdc45 and GINS have been shown to preferentially associate with Mcms during S phase (15, 16, 38). Furthermore, the bob-1 mutant, an allele of Mcm5 in S. cerevisiae, suppresses the requirement for the Cdc7 kinase (39), and it is tempting to speculate that this mutation bypasses a modification that is essential for CMG complex assembly.

The exclusive assembly of Cdc45 and GINS with Mcm2–7 at a prereplication complex just before and during DNA synthesis would also clarify why only a small percentage of the total Cdc45, GINS, and Mcm2–7 proteins in our starting nuclear extract are found in the complex (see Fig. 7). A free pool of Cdc45 and GINS proteins may be required for activation of replication origins throughout S phase. Only a small percentage of Mcm proteins in the nucleus are in the CMG complex; therefore, most Mcm proteins may not be located at sites of DNA unwinding. The vast excess of Mcm2–7 in the extracts that is not in the CMG complex is consistent with reports that most Mcm proteins do not localize to sites of DNA replication in metazoans (40, 41).

Taking further the notion that the helicase activity of the CMG complex is provided by Mcm2–7, what role might Cdc45 and GINS play in this function? It is possible that the CMG complex forms only at preinitiation sites and that, concomitant with this assembly, a specific set of posttranslational modifications of the initiation factors activates the helicase activity. Further dissection of the modification patterns of the proteins of the CMG complex and an understanding of how the complex is assembled will answer these questions. Apart from specific modifications, a simple notion would be that Cdc45 and/or GINS induces or stabilizes a conformational change in the Mcm2–7 hexamer, serving as a molecular switch that converts an inactive helicase to an active form. Cdc45 and GINS may thus be a part of the actual helicase machinery, with, for example, Cdc45 possibly serving as a wedge in the recently proposed “plowshare” model for helicase activity (1). A second, nonexclusive possibility is that Cdc45 and GINS associate with the Mcm2–7 helicase complex for purposes of coordination with DNA repair factors. Cdc45 has been shown to associate with the checkpoint proteins Mrc1 and Tof1 during S phase (42, 43), and we stress that our stringent purification methods might dislodge the Drosophila homolog of Claspin/Mrc1 from the CMG complex. The cell's central replicative helicase may only be activated when the factors that can coordinate helicase function with checkpoint proteins are fully engaged.

CMG as a Helicase.

The data presented in this study indicate that a purified Mcm2–7-containing complex has DNA helicase activity. The question may be raised of whether the Mcm2–7 proteins of the CMG complex are functioning as a helicase or whether the CMG complex either dissociates to a Mcm4,6,7 subcomplex or copurifies with an unrecognized helicase.

Although we cannot formally disprove either of these possibilities, our data suggest that both of these possibilities are unlikely. The CMG complex is stable through many chromatography steps and high-salt conditions, and we believe that it is unlikely that the complex would dissociate during the gentle conditions of the helicase assays. In addition, if it did dissociate, then the free Mcm2, Mcm3, and Mcm5 that would result from the dissociation would be expected to inhibit the Mcm4,6,7 activity. The second possibility, that there is an unrecognized helicase complex that copurifies with the CMG complex, is also unlikely. The depletion experiments in Fig. 3D indicate that the observed helicase activity is tightly associated with the CMG complex. Examination of the CMG complex purified by anti-Cdc45, anti-FLAG/anti-Cdc45, or anti-Psf2 chromatography (Figs. 1 and and2)2) suggests that CMG does not have any unidentified binding partners that could provide helicase activity. Hence, the best explanation of the data shown here is that the observed helicase activity is manifest from the complex itself.

Molecular Architecture of the CMG Complex.

Electron microscope images of Mcm2–7 and GINS complexes individually have shown that both complexes form ring-shaped structures (28, 44). These findings suggest that the two rings may stack on top of each other to form a common central channel that could surround ssDNA or dsDNA. Fig. 5B shows a speculative model of the molecular architecture of the CMG complex. Double hexamers of the archaeal Mcm rings have been observed (7, 45), and, by analogy, one might expect the Drosophila Mcm2–7 to form such double hexamer structures. However, our present model posits that the active helicase contains a six-member (Mcm2–7) and four-member (GINS) ring ensemble. The model of one Mcm2–7 hexamer per CMG complex is based on gel filtration data, which show that the CMG complex migrates at the same position as thyrogloblulin (669 kDa) on both Superdex-200 (Fig. 1C) and Superose 6 (see Fig. 10, which is published as supporting information on the PNAS web site) columns, suggesting that the mass of the CMG complex is close to 700 kDa. The calculated molecular mass of a complex containing one Mcm2–7 hexamer, one GINS tetramer, and one Cdc45 molecule is 708 kDa.

A recent study (6) showed that Mcm2, Mcm5, Mcm7, Sld5, and Cdc45 localize to sites of DNA unwinding on a plasmid in Xenopus extracts, a result that is consistent with the present data. In the study (6), unwinding was uncoupled from DNA synthesis, and some fraction of DNA replication fork proteins were associated with the unwinding activity. These researchers suggested that the entire set of Mcm2–7, Cdc45, and the GINS complex were part of a molecular machine that they referred to as the “unwindosome.” The term unwindosome may perhaps refer to a very large collection of proteins yet to be identified that comprise and associate with the DNA unwinding machinery. In fact, another recent study (43) shows that a large number of S. cerevisiae proteins associate with Sld5 and Mcm4 in a high-molecular-weight complex referred to as the “replisome progression complex.” In this study, we show that Cdc45, Mcm2–7, and GINS form a biochemically discrete complex, and we propose that the CMG complex per se forms the core of helicase activity.

Materials and Methods

CMG Complex Purification.

The experimental details of CMG complex purification are described in the supporting information.


Antibodies against Drosophila Cdc45, Sld5, Psf1, Psf2, and Psf3 proteins were raised by cloning the respective full-length genes into pQE-30 (Qiagen, Valencia, CA) and expressing His-tagged versions of each protein in the Escherichia coli strain XA-90. All proteins were solubilized with 6 M Gu-HCl and purified by binding to Ni-nitrilotriacetic acid resin (Qiagen), followed by imidazole elution. For each protein, two New Zealand White rabbits were injected with the respective purified protein mixed with Ribi adjuvant (Corixa, Seattle). Anti-Cdc45 antibodies were affinity-purified by standard methods by using maltose-binding protein-linked Cdc45 protein as the target protein. Antibodies against Mcm2, Mcm4, and Mcm5 were a generous gift from T. T. Su (University of Colorado, Boulder).


For antibody resins, antibodies were mixed with protein A Sepharose CL-4B beads (Amersham Pharmacia) and cross-linked by standard methods. IPs were performed by mixing antibody/protein A resins with protein samples overnight, followed by extensive washing of the resin. Except where noted in the text, bound proteins were eluted from antibody/protein A beads by elution with glycine buffer (pH 2.5) and immediately neutralized with 1 M Tris·Cl (pH 8.0). For Cdc45 peptide B elutions, the anti-Cdc45 peptide B antibody resins were incubated for 10 min with a buffer containing 50 mM Hepes (pH 7.58), 10 mM magnesium acetate, 50 mM sodium acetate, 10% glycerol, 0.25 mg/ml insulin (Sigma), and 200 μg/ml Cdc45 peptide B.

FLAG-Mcm6 Material Purification.

Extract was prepared from 0- to 12-h embryos of flies expressing FLAG-Mcm6. The extract was mixed with anti-FLAG M2 resin (Sigma) overnight and washed extensively with buffer containing 1 M KCl. Material was eluted from the anti-FLAG resin by competition with “Cdc45 buffer” (see the CMG complex purification protocol in the supporting information) containing 100 μg/ml FLAG peptide.

Mcm4,6,7 Purification.

Purified FLAG-Mcm6 material was loaded onto a Mono Q column and gradient-eluted, which separates Mcm4,6,7 complexes from Mcm2,4,6,7 complexes. The peak of Mcm4,6,7 elution is from 280 to 310 mM KCl, and the Mcm2,4,6,7 peak is from 325 to 380 mM KCl.

Mass Spectroscopy.

2D analysis of total protein eluate from the anti-Cdc45 IP was performed by the Proteomics/Mass Spectrometry Facility (University of California, Berkeley). Individual protein bands were identified by AmProx (Carlsbad, CA).


Cdc45 peptides A and B were prepared by GenScript (Piscataway, NJ).

Helicase Substrates and Assays.

The experimental details of the helicase substrates and assays are described in the supporting information.

RNAi and FACS Analysis.

dsRNA synthesis, RNA transfection, and total cellular RNA purification for Cdc45, Sld5, Psf1, Psf2, and Psf3 were performed as described in ref. 46. RNAi-treated cells were stained with propidium iodide for FACS analysis.

Supplementary Material

Supporting Information:


We dedicate this paper to the memory of our mentor, friend, and colleague, Nicholas Cozzarelli. We thank Brian Calvi (University of Pennsylvania, Philadelphia) for the FLAG-Mcm6 strain of Drosophila, Tin Tin Su for anti-Mcm antibodies, and Sue Cotterill (St. Georges, University of London, London) for anti-Cdc45 antibodies used in the initial stages of this work. This work was supported by National Institutes of Health Grant CA R37-30490.


RNA interference.


Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.


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