Isolation of the Cdc45͞Mcm2–7͞GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork

Stephen E. Moyer, Peter W. Lewis, and Michael R. Botchan*

Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720

Edited by Bruce W. Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved May 22, 2006 (received for review March 23, 2006) The Cdc45 plays a critical but poorly understood role in the (28). Many of these interactions may be important but transient initiation and elongation stages of eukaryotic DNA replication. To or indirect and may obscure a core function or complex within study Cdc45’s function in DNA replication, we purified Cdc45 which Cdc45 works. protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found Results that the protein exists in a stable, high-molecular-weight complex Purification and Identification of the Cdc45͞Mcm2–7͞GINS (CMG) with the Mcm2–7 hexamer and the GINS tetramer. The purified Complex. A protocol to purify a high-molecular-weight complex Cdc45͞Mcm2–7͞GINS complex is associated with an active ATP- containing the Cdc45 protein from Drosophila embryo extracts dependent DNA helicase function. RNA interference knock-down is outlined in Fig. 1A (see Materials and Methods and see also experiments targeting the GINS and Cdc45 components establish Table 1 and Supporting Text, which are published as supporting that the are required for the S phase transition in Dro- information on the PNAS web site, for additional details). Final sophila cells. The data suggest that this complex forms the core purification used immunoprecipitation (IP) of Cdc45 protein helicase machinery for eukaryotic DNA replication. from the peak Cdc45-containing fractions of the Mono Q ͉ column and elution from the antibody resin with buffer (pH 2.5). Drosophila ATPase Before elution, the material in the IP was extensively washed with buffer containing 1 M KCl and treated with ethidium lthough in recent years there has been significant progress bromide and MNase to remove any contaminating DNA. As Ain our understanding of the molecular mechanisms of shown in Fig. 1B and Fig. 6, which is published as supporting eukaryotic DNA replication, the identity of the primary activity information on the PNAS web site, Cdc45 coimmunoprecipitates that unravels duplex DNA at the growing fork has remained with 10 other major proteins. The identity of the copurifying unclear. Based on a range of indirect data, there is widespread proteins was determined by two methods: (i) mass spectroscopy support for the idea that the Mcm2–7 hexamer constitutes part of individual protein bands cut out from the gel (see List 1, which of the replicative helicase (1). Each of the is essential, and is published as supporting information on the PNAS web site) five of six Mcm2–7 proteins have been shown to be required for and (ii) immunoblotting of the eluate with protein-specific both DNA replication initiation and elongation in vivo in Sac- antibodies. charomyces cerevisiae (2, 3). Assays with Xenopus cell-free ex- The 10 binding partners of Cdc45 were found to be the six tracts have shown that all Mcm2–7 proteins are required for chromosomal DNA unwinding and that Mcm2, Mcm5, and proteins of the Mcm2–7 complex and CG14549, CG9187, Mcm7 localize to sites of DNA unwinding on a plasmid (4–6). CG18013, and CG2222, which BLAST searches revealed are the The primary structure of the proteins places each in the helicase Drosophila homologs of the four members of the GINS complex supergroup of the AAAϩ family, and archaeal forms do show (Sld5, Psf1, Psf2, and Psf3, respectively) (Fig. 1B). We also robust helicase function (7–9). Curiously, the purified Mcm2–7 performed mass spectroscopy analysis on the total protein pool complex has not displayed helicase activity in vitro; in contrast, obtained after an anti-Cdc45 IP from the Cdc45-containing the subcomplex of Mcm4, Mcm6, and Mcm7 does (10–12). DEAE fractions from the protocol described in Fig. 1A (see Moreover, the activity of this subcomplex is inhibited by Mcm2, Table 2, which is published as supporting information on the Mcm3, or Mcm5 (10, 11, 13, 14). Intricate models involving PNAS web site). The only DNA replication proteins identified continuous assembly and rearrangements of Mcm proteins at the by this method were also Cdc45, Mcm2–7, and GINS. replication fork could somehow accommodate these findings. A Sld5, the founding member of the GINS complex, was first simpler hypothesis posits that other proteins are required to uncovered in a S. cerevisiae synthetic lethal screen with Dpb11 activate the Mcm 2–7 complex for helicase function. (29). Dpb11 was itself genetically isolated in a suppressor screen The protein Cdc45 has emerged as a pivotal factor in the G1 for a mutant in a subunit of DNA ␧ (30). The Psf to S phase transition and as a possible helicase cofactor (4, 6, 15, genes are each partners of Sld5 and are required for DNA 16). CDC45 is an essential product in yeast (17) and synthesis in yeast and in Xenopus cell-free DNA replication engages proteins assembled in the prereplication complex at extracts (28, 29). The GINS proteins form a tetramer, and it was origin sites on the DNA during S phase as origins are activated recently shown that Sld5 localizes to sites of DNA unwinding on (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 Conflict of interest statement: No conflicts declared. (4). There is a conserved genetic interaction between Cdc45 and This paper was submitted directly (Track II) to the PNAS office. the Mcms; specific mutations in these genes can either suppress Freely available online through the PNAS open access option. or show synthetic lethality with the other (19–21). By coimmu- Abbreviations: CMG, Cdc45͞Mcm2–7͞GINS; IP, immunoprecipitation; RNAi, RNA interference. ͞ noprecipitation immunoblot experiments, Cdc45 has been *To whom correspondence should be addressed at: Department of Molecular and Cell shown to associate with a variety of other DNA replication Biology, University of California, 16 Barker Hall, Berkeley, CA 94720. E-mail: mbotchan@ proteins, including DNA polymerase ␣ (22, 23), various Mcm2–7 berkeley.edu. proteins (16, 23, 24), Mcm10 (25), Orc2 (26), Sld3 (27), and Sld5 © 2006 by The National Academy of Sciences of the USA

10236–10241 ͉ PNAS ͉ July 5, 2006 ͉ vol. 103 ͉ no. 27 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602400103 Downloaded by guest on September 27, 2021 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 chromatogra- phy. (Right) From the material first purified by the anti-FLAG chromatography, an anti-Cdc45 IP and a control (rabbit IgG) IP were performed. The anti-Cdc45 immunoprecipitated material shown here is enriched for Cdc45 Ϸ70-fold over the purified FLAG-Mcm6 material (i.e., Ϸ700 ␮l of the material in Left was used to obtain 10 ␮l of the material in Right). Both gels were stained with Deep Purple. (B) Peak Cdc45-containing fractions from the Mono Q step were precipitated with Fig. 1. Purification of the CMG complex. (A) Purification schematic. See the anti-Psf2 antibodies and control antibodies (anti-FLAG), and the eluates were supporting information for additional details. (B) Material eluted from the stained with Deep Purple. anti-Cdc45 IP after the Mono Q step was separated by SDS͞PAGE and visual- ized by Deep Purple stain. For improved resolution of individual bands, the image is composed of the eluate separated by two different acrylamide gels: To further explore the notion that a discrete 11-member CMG 9% (top of gel to IgG) and 12% (IgG to bottom of gel). The complete lane of complex exists, we embarked on independent purification meth- each gel used for this image is shown in Fig. 6. All subsequent gels in this study ods. We prepared 0- to 12-h embryo extracts from Drosophila are 10% acrylamide and contain all proteins in one complete lane. (C)Atthe expressing a functional, FLAG-tagged version of Mcm6 (32). A Superdex-200 step shown in A, each fraction was subjected to an anti-Cdc45 single-step affinity purification of FLAG-Mcm6 from these flies IP, and the individual eluates were separated on SDS͞PAGE and immunoblot- ted with antibodies against the indicated proteins. The antibodies have captures mainly FLAG-Mcm6 in complex with Mcm2, Mcm4, variable staining intensity; the ratio of all CMG proteins to each other in the and Mcm7 (Fig. 2A Left). This result was anticipated because high-molecular-weight fractions is the same (see Figs. 1B, 2, and 6). subcomplexes of the Mcm proteins are in vast (Ͼ100-fold) excess to the high-molecular-weight complex we had uncovered. IP of Cdc45 from the purified FLAG-Mcm6 material yielded the a plasmid in Xenopus egg extracts (6), suggesting that all four complete CMG complex (Fig. 2A Right). In another set of GINS proteins may be present in a DNA helicase complex. experiments, we fractionated embryo extract, following the Examination of the Superdex-200 column fractions from Fig. high-molecular-weight Cdc45 through the Mono Q step as in Fig. 1A shows that the majority of the Cdc45 protein in these extracts 1A, and with antibodies directed against Mcm5, Sld5, Psf2, and exists as a free, low-molecular-weight pool (see Fig. 1C and Fig. Psf3, asked whether the individual reagents would in turn BIOCHEMISTRY 7, which is published as supporting information on the PNAS precipitate the proteins identified by previous methods. Fig. 2B web site). However, only discrete high-molecular-weight frac- shows a Deep Purple stain of the material eluted from the tions contain a complex that shows an interaction among Cdc45, anti-Psf2 IP. Cdc45 and the complete Mcm2–7 complex coim- the GINS members, and the Mcm proteins. Fig. 1C shows an munoprecipitated with the GINS complex, and immunoblots of immunoblot of the material eluted from an anti-Cdc45 IP from each Superdex-200 chromatography fraction. The size of the the material eluted from each IP (see Fig. 8, which is published complex as estimated from the elution position on the column as supporting information on the PNAS web site) confirmed the closely matches the sum of the individual components (see tight association of each of the 11 members of this complex. Discussion). We were not able to reform the high-molecular- Based on these multiple lines of evidence, we conclude that weight complex by mixing of lower molecular-weight fractions, Cdc45, Mcm2–7, and GINS form a stable, high-molecular-weight which contain an excess of the components (see Fig. 7). We biochemical unit, which we refer to as the CMG complex. An estimate that Ϸ5% of the total Cdc45 and GINS proteins and immediate question was whether the complex contained an Ϸ1% of the total Mcm proteins in our extracts are in this active form of the hypothetical Mcm2–7 helicase. complex. These results suggest a role for specific protein mod- ifications that would allow for such associations and would Purification of the CMG Complex by Peptide Elution from an Affinity support a previous report that the Cdc45 protein does not Resin. We attempted to purify the CMG complex to homogeneity interact with individual Mcm2–7 proteins (31). by conventional chromatography but found that the only bio-

Moyer et al. PNAS ͉ July 5, 2006 ͉ vol. 103 ͉ no. 27 ͉ 10237 Downloaded by guest on September 27, 2021 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 from each Mono Q fraction. (Middle) Quantitation of the percentage of primers displaced from duplex DNA in each helicase reaction. (Bottom) Immunoblot with antibodies against indicated proteins of the anti-Cdc45 peptide B-purified material from each Mono Q fraction. The Cdc45 protein peak in fractions 12–15 is monomeric Cdc45 that has copurified with the CMG complex until this chromatography step. (Left) Deep Purple stain of anti-Cdc45 peptide B-purified material from fraction 20. (D)(Left) Immunoblots of material that has been immunodepleted with anti-Psf2, anti-Mcm5, or control antibodies. (Right) PhosphorImager picture of helicase assays with protein purified by anti-Cdc45 peptide B IPs from material previously depleted with anti-Psf2, anti-Mcm5, or control antibodies. (E)(Left) Helicase assays with material mock-eluted or Cdc45 peptide B-eluted from anti-Cdc45 peptide B IPs from purified FLAG-Mcm6 material. (Center) Helicase assays with and without ATP with CMG complex isolated from purified FLAG-Mcm6 material. (Right) Helicase assays with CMG complex purified as in A and with varying concentrations of ATP.

chemical step that accomplished this purification was an anti- complex according to the procedure outlined in Fig. 3A and Cdc45 affinity resin. Thus, to assay the CMG complex for asked whether the purified material had helicase activity. The helicase activity, we sought to release the CMG complex from substrate used contains a 40-bp duplex region with a short tail antibodies with a gentle elution method that did not abolish any annealed to a single-stranded circle (Fig. 3B). Fig. 3C shows that intrinsic enzymatic function. We searched for peptide-specific helicase activity peaks with the protein peak of the purified anti-Cdc45 antibodies and explored releasing the intact CMG CMG complex. Fig. 3C Left shows a Deep Purple stain of the complex from the anti-Cdc45 antibody beads by peptide elution material purified by this protocol from Mono Q fraction 20, (see Fig. 9A, which is published as supporting information on the indicating the presence of the complete CMG complex. PNAS web site). We selected two peptides from a region of Immunodepletion of column fractions with specific antisera Cdc45 that is hydrophilic and predicted to have no secondary raised to recombinant proteins showed that the activity we had structure (see Fig. 9B). Purified antibodies specific to peptide B can immunoprecipitate Cdc45 protein and release Cdc45 when assayed above was directly associated with the CMG complex. challenged with excess free peptide B in a neutral buffer (see Fig. We pooled material from Mono Q fractions 18–21, divided it 9C). Importantly, the anti-Cdc45 peptide B antibodies can also into three portions, and then immunodepleted it with specific bind and release Cdc45 protein that is in the CMG complex (Fig. antibodies. The antibody beads were all prepared in the same 3C), indicating that this region is on the surface of the Cdc45 buffer to minimize the possibility of a nonspecific inhibitor being protein and that it is exposed on both free Cdc45 protein and present with any of the antibody resins. Fig. 3D Left examines the Cdc45 protein in the CMG complex. extent of depletion in the material treated with control, anti- Psf2, or anti-Mcm5 antibodies. We were able to immunodeplete Identification of CMG-Associated Helicase Activity. We used the Psf2 protein to levels below detection and to at least 50% of the anti-Cdc45 peptide B-specific antibodies to purify the CMG starting level for Mcm5 protein. From the immunodepleted

10238 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602400103 Moyer et al. Downloaded by guest on September 27, 2021 Fig. 5. Cdc45 and GINS proteins are required for normal S phase progression. (A)(Left) Drosophila Kc tissue culture cells were treated with Ϸ500 bp of dsRNA directed against Cdc45, Sld5, Psf1, Psf2, Psf3, or nonspecific (NS) RNA. After 8 days of RNAi treatment, cells were harvested; FACS profiles are shown. Fig. 4. CMG directionality and . (A)(Left) Cartoon of procedure The x axis is arbitrary fluorescence units, and the y axis is the number of cells for preparing substrates for directionality assays. A 48-mer primer was an- (0–128). (Right) Cell cycle distribution of RNAi-treated cells. (B) A model of the 32 Ј nealed to m13mp18 ssDNA plasmid and then labeled with P at the 5 end CMG complex. Green, Mcm2–7; blue, GINS; yellow, Cdc45. with T4 polynucleotide kinase and [␥-32P]ATP or at the 3Ј end with T7 DNA polymerase and [␣-32P]dGTP. The duplex region was digested with SmaI, creating a linear plasmid with ssDNA in the middle and duplex DNA at both peptide B elution and indicates that the detected helicase in ends (the 5Ј labeled primer is 20 bases, and the 3Ј labeled primer is 28 bases). The radiolabeled primer can only be approached on ssDNA from one direc- these assays is associated with the CMG complex. We have found tion, thereby allowing determination of the directionality of helicase move- in our various preparations of the CMG complex that the ment on DNA. (Right) PhosphorImager picture of CMG complex tested with helicase activity is ATP-dependent (Fig. 3E Center) and satu- both directionality substrates. (B) The primer in Fig. 3B was extended by the rates with 1 mM nucleotide (Fig. 3E Right). DNA polymerase of T7 (Sequenase), and the heterogeneous products created Two substrates (Fig. 4A Left) were used to test the direction- substrates for helicase assays. A PhosphorImager picture of DNA substrates ality of the CMG complex tracking movement on DNA. As after incubation in helicase assays with increasing amounts of CMG or indicated by the displacement of the 5Ј labeled substrate in Fig. Mcm4,6,7 complexes is shown. For measuring the lengths of the duplex DNA 4A Right, the CMG complex translocates on DNA in a 3Ј–5Ј in the substrates, a dsDNA ladder was labeled with 32P and separated on the gel in either native or boiled form. direction. We have also determined that the CMG complex will displace a substrate identical to that in Fig. 3B, except with a 3Ј 30T tail instead of a 5Ј tail (data not shown). Thus, the CMG material, we attempted to purify the CMG complex with anti- complex can load onto helicase substrates with a gapped single Cdc45 peptide B beads, and the respective eluted proteins were strand (Fig. 4A)or5Ј or 3Ј overhangs, like Mcm4,6,7 (10, 33). tested for helicase activity. Fig. 3D Right shows that the Psf2- To assay the processivity of the CMG helicase, we annealed depleted material contains no Cdc45-associated helicase activ- the primer used in Fig. 3B to m13mp18 ssDNA plasmid and ity, and the Mcm5-depleted material has reduced Cdc45- extended the primer with T7 DNA polymerase to generate a associated helicase activity, consistent with the reduction, but population of duplex primer–plasmid regions of various and not complete removal, of Mcm5 protein from the starting heterogeneous lengths. Fig. 4B shows the results of these helicase protein pool. The material depleted with control antisera assays with titrations of the CMG complex, and we conclude that BIOCHEMISTRY showed robust Cdc45-associated activity. These results demon- the complex can work processively for many hundreds of base strate that the helicase activity observed in Fig. 3C is associated pairs. From analysis of side-by-side comparisons with the Dro- with the CMG complex and that Psf2 and Mcm5 are components sophila Mcm4,6,7 subcomplex, we conclude that the CMG of the active helicase complex. complex is as processive as the subcomplex and perhaps more Helicase activity was also identified in CMG complex isolated active at lower levels of protein. from purified FLAG-Mcm6 material (Fig. 3E Left and Center). Anti-Cdc45 peptide B resin was mixed with purified FLAG- In Vivo Requirement of Cdc45 and GINS. The four proteins of the Mcm6-containing complexes, the resin was washed extensively, GINS complex are required for cell cycle progression in yeast and bound CMG complexes were eluted with a neutral buffer (29), but parallel in vivo or cell-based data have not been containing Cdc45 peptide B. To ensure that any observed reported for most GINS members in metazoans. We depleted helicase activity in these preparations was not from residual Cdc45 or the GINS complex members from Drosophila Kc tissue Mcm4,6,7 complex that may have not been completely washed culture cells by RNA interference (RNAi) and examined the cell from the resin after binding CMG to the anti-Cdc45 peptide B cycle distribution of the treated cells. Loss of Cdc45 or any one beads, we mock-eluted with buffer containing FLAG peptide of the GINS complex members results in a significant impair- before eluting with buffer containing Cdc45 peptide B. Fig. 3E ment of cell cycle progression and an accumulation of cells in G1 Left shows helicase assays with the material eluted from the and S phase (Fig. 5A). These results support a recent report that anti-Cdc45 peptide B resin by the mock elution or the Cdc45 Psf1 is required for embryo development and cell proliferation

Moyer et al. PNAS ͉ July 5, 2006 ͉ vol. 103 ͉ no. 27 ͉ 10239 Downloaded by guest on September 27, 2021 in mice (34) and indicate an in vivo requirement for GINS during CMG as a Helicase. The data presented in this study indicate that S phase in metazoans. a purified Mcm2–7-containing complex has DNA helicase ac- tivity. The question may be raised of whether the Mcm2–7 Discussion proteins of the CMG complex are functioning as a helicase or We have provided evidence that Cdc45 exists in a stable whether the CMG complex either dissociates to a Mcm4,6,7 biochemical unit with the Mcm2–7 and GINS complexes and subcomplex or copurifies with an unrecognized helicase. that this large complex has associated with it an ATP-dependent Although we cannot formally disprove either of these possi- helicase activity. The evidence that the Mcm2–7 complex is bilities, our data suggest that both of these possibilities are responsible for this activity (as opposed to another helicase or a unlikely. The CMG complex is stable through many chromatog- subset of the Mcm proteins) is not definitive, but it is an raphy steps and high-salt conditions, and we believe that it is attractive hypothesis. Reconstitution of the complex from re- unlikely that the complex would dissociate during the gentle combinant proteins will be the next step in testing this notion. conditions of the helicase assays. In addition, if it did dissociate, The identification of the CMG complex and its associated then the free Mcm2, Mcm3, and Mcm5 that would result from helicase activity supports previous reports that implicate the the dissociation would be expected to inhibit the Mcm4,6,7 Mcms, Cdc45, and GINS in unwinding (4–6), and activity. The second possibility, that there is an unrecognized it begins to provide a molecular model for the mechanism of helicase complex that copurifies with the CMG complex, is also DNA unwinding at the eukaryotic DNA replication fork. unlikely. The depletion experiments in Fig. 3D indicate that the observed helicase activity is tightly associated with the CMG Formation of the CMG Complex. Cdc45 and GINS first associate complex. Examination of the CMG complex purified by anti- with replication origins at the G1 to S phase transition after the Cdc45, anti-FLAG͞anti-Cdc45, or anti-Psf2 chromatography activation of the cyclin-dependent kinase (Cdk) and Cdc7 pro- (Figs. 1 and 2) suggests that CMG does not have any unidentified tein kinases (16, 35), and it is possible that phosphorylation of binding partners that could provide helicase activity. Hence, the ͞ one or more of the CMG complex members by Cdk and or Cdc7 best explanation of the data shown here is that the observed may promote Cdc45 and GINS association with Mcm2–7. Mcm helicase activity is manifest from the complex itself. proteins have been shown to be phosphorylated at the G1 to S phase transition (36, 37), and Cdc45 and GINS have been shown Molecular Architecture of the CMG Complex. Electron microscope to preferentially associate with Mcms during S phase (15, 16, 38). images of Mcm2–7 and GINS complexes individually have shown Furthermore, the bob-1 mutant, an allele of Mcm5 in S. cerevi- that both complexes form ring-shaped structures (28, 44). These siae, suppresses the requirement for the Cdc7 kinase (39), and it findings suggest that the two rings may stack on top of each other is tempting to speculate that this mutation bypasses a modifi- to form a common central channel that could surround ssDNA cation that is essential for CMG complex assembly. or dsDNA. Fig. 5B shows a speculative model of the molecular The exclusive assembly of Cdc45 and GINS with Mcm2–7 at architecture of the CMG complex. Double hexamers of the a prereplication complex just before and during DNA synthesis archaeal Mcm rings have been observed (7, 45), and, by analogy, would also clarify why only a small percentage of the total Cdc45, one might expect the Drosophila Mcm2–7 to form such double GINS, and Mcm2–7 proteins in our starting nuclear extract are hexamer structures. However, our present model posits that the found in the complex (see Fig. 7). A free pool of Cdc45 and GINS active helicase contains a six-member (Mcm2–7) and four- proteins may be required for activation of replication origins throughout S phase. Only a small percentage of Mcm proteins in member (GINS) ring ensemble. The model of one Mcm2–7 the nucleus are in the CMG complex; therefore, most Mcm hexamer per CMG complex is based on gel filtration data, which proteins may not be located at sites of DNA unwinding. The vast show that the CMG complex migrates at the same position as excess of Mcm2–7 in the extracts that is not in the CMG complex thyrogloblulin (669 kDa) on both Superdex-200 (Fig. 1C) and is consistent with reports that most Mcm proteins do not localize Superose 6 (see Fig. 10, which is published as supporting to sites of DNA replication in metazoans (40, 41). information on the PNAS web site) columns, suggesting that the Taking further the notion that the helicase activity of the mass of the CMG complex is close to 700 kDa. The calculated CMG complex is provided by Mcm2–7, what role might Cdc45 molecular mass of a complex containing one Mcm2–7 hexamer, and GINS play in this function? It is possible that the CMG one GINS tetramer, and one Cdc45 molecule is 708 kDa. complex forms only at preinitiation sites and that, concomitant A recent study (6) showed that Mcm2, Mcm5, Mcm7, Sld5, and with this assembly, a specific set of posttranslational modifica- Cdc45 localize to sites of DNA unwinding on a plasmid in tions of the initiation factors activates the helicase activity. Xenopus extracts, a result that is consistent with the present data. Further dissection of the modification patterns of the proteins of In the study (6), unwinding was uncoupled from DNA synthesis, the CMG complex and an understanding of how the complex is and some fraction of DNA replication fork proteins were assembled will answer these questions. Apart from specific associated with the unwinding activity. These researchers sug- modifications, a simple notion would be that Cdc45 and͞or gested that the entire set of Mcm2–7, Cdc45, and the GINS GINS induces or stabilizes a conformational change in the complex were part of a molecular machine that they referred to Mcm2–7 hexamer, serving as a molecular switch that converts an as the ‘‘unwindosome.’’ The term unwindosome may perhaps inactive helicase to an active form. Cdc45 and GINS may thus be refer to a very large collection of proteins yet to be identified that a part of the actual helicase machinery, with, for example, Cdc45 comprise and associate with the DNA unwinding machinery. In possibly serving as a wedge in the recently proposed ‘‘plowshare’’ fact, another recent study (43) shows that a large number of S. model for helicase activity (1). A second, nonexclusive possibility cerevisiae proteins associate with Sld5 and Mcm4 in a high- is that Cdc45 and GINS associate with the Mcm2–7 helicase molecular-weight complex referred to as the ‘‘ pro- complex for purposes of coordination with DNA repair factors. gression complex.’’ In this study, we show that Cdc45, Mcm2–7, Cdc45 has been shown to associate with the checkpoint proteins and GINS form a biochemically discrete complex, and we Mrc1 and Tof1 during S phase (42, 43), and we stress that our propose that the CMG complex per se forms the core of helicase stringent purification methods might dislodge the Drosophila activity. homolog of Claspin͞Mrc1 from the CMG complex. The cell’s central replicative helicase may only be activated when the Materials and Methods factors that can coordinate helicase function with checkpoint CMG Complex Purification. The experimental details of CMG com- proteins are fully engaged. plex purification are described in the supporting information.

10240 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602400103 Moyer et al. Downloaded by guest on September 27, 2021 Antibodies. Antibodies against Drosophila Cdc45, Sld5, Psf1, Psf2, (see the CMG complex purification protocol in the supporting and Psf3 proteins were raised by cloning the respective full-length information) containing 100 ␮g͞ml FLAG peptide. genes into pQE-30 (Qiagen, Valencia, CA) and expressing His- tagged versions of each protein in the Escherichia coli strain XA-90. Mcm4,6,7 Purification. Purified FLAG-Mcm6 material was All proteins were solubilized with 6 M Gu-HCl and purified by loaded onto a Mono Q column and gradient-eluted, which binding to Ni-nitrilotriacetic acid resin (Qiagen), followed by imi- separates Mcm4,6,7 complexes from Mcm2,4,6,7 complexes. dazole elution. For each protein, two New Zealand White rabbits The peak of Mcm4,6,7 elution is from 280 to 310 mM KCl, and were injected with the respective purified protein mixed with Ribi the Mcm2,4,6,7 peak is from 325 to 380 mM KCl. adjuvant (Corixa, Seattle). Anti-Cdc45 antibodies were affinity- purified by standard methods by using maltose-binding protein- Mass Spectroscopy. 2D analysis of total protein eluate from the linked Cdc45 protein as the target protein. Antibodies against anti-Cdc45 IP was performed by the Proteomics͞Mass Spec- Mcm2, Mcm4, and Mcm5 were a generous gift from T. T. Su trometry Facility (University of California, Berkeley). Individual (University of Colorado, Boulder). protein bands were identified by AmProx (Carlsbad, CA).

IPs. For antibody resins, antibodies were mixed with protein A Peptides. Cdc45 peptides A and B were prepared by GenScript Sepharose CL-4B beads (Amersham Pharmacia) and cross- (Piscataway, NJ). linked by standard methods. IPs were performed by mixing ͞ antibody protein A resins with protein samples overnight, fol- Helicase Substrates and Assays. The experimental details of the lowed by extensive washing of the resin. Except where noted in helicase substrates and assays are described in the supporting ͞ the text, bound proteins were eluted from antibody protein A information. beads by elution with glycine buffer (pH 2.5) and immediately ⅐ neutralized with 1 M Tris Cl (pH 8.0). For Cdc45 peptide B RNAi and FACS Analysis. dsRNA synthesis, RNA transfection, and elutions, the anti-Cdc45 peptide B antibody resins were incu- total cellular RNA purification for Cdc45, Sld5, Psf1, Psf2, and bated for 10 min with a buffer containing 50 mM Hepes (pH Psf3 were performed as described in ref. 46. RNAi-treated cells 7.58), 10 mM magnesium acetate, 50 mM sodium acetate, 10% were stained with propidium iodide for FACS analysis. glycerol, 0.25 mg͞ml insulin (Sigma), and 200 ␮g͞ml Cdc45 peptide B. We dedicate this paper to the memory of our mentor, friend, and colleague, Nicholas Cozzarelli. We thank Brian Calvi (University of FLAG-Mcm6 Material Purification. Extract was prepared from 0- to Pennsylvania, Philadelphia) for the FLAG-Mcm6 strain of Drosophila, 12-h embryos of flies expressing FLAG-Mcm6. The extract was Tin Tin Su for anti-Mcm antibodies, and Sue Cotterill (St. Georges, mixed with anti-FLAG M2 resin (Sigma) overnight and washed University of London, London) for anti-Cdc45 antibodies used in the extensively with buffer containing 1 M KCl. Material was eluted initial stages of this work. This work was supported by National Institutes from the anti-FLAG resin by competition with ‘‘Cdc45 buffer’’ of Health Grant CA R37-30490.

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