Title: The Regulated Association of Cdt1 with Minichromosome Maintenance Proteins and Cdc6 in Mammalian Cells
Abstract: 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. Here we demonstrate that Cdc6 and Cdt1 are 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 G1, is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and we provide evidence 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. 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. Here we demonstrate that Cdc6 and Cdt1 are 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 G1, is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and we provide evidence 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. Eukaryotic chromosomes undergo a highly regulated preparation process during the G1 phase of the cell cycle in order to allow DNA replication and the initiation of S phase. An early step in laying the groundwork for DNA replication is the formation of prereplication complexes at multiple chromosomal origins. Prereplication complexes are generated by the ordered addition of the six-subunit origin recognition complex (ORC), 1The abbreviations used are: ORC, origin recognition complex; Mcm, minichromosome maintenance; GST, glutathione S-transferase; CMV, cytomegalovirus; MOI, multiplicity of infection. the Cdc6 and Cdt1 proteins, and the six-subunit minichromosome maintenance complex (Mcm) to the presumptive origin. Individual members of the prereplication complex were initially identified by genetic screens in yeast or by biochemical purification from Xenopus laevis egg extracts, and orthologs of each have been identified in all eukaryotic species examined. Each factor has been shown to be strictly required for DNA replication, and the molecular functions of many of these proteins have been characterized (for reviews, see Refs. 1Ritzi M. Knippers R. Gene (Amst.). 2000; 245: 13-20Crossref PubMed Scopus (47) Google Scholar, 2Takisawa H. Mimura S. Kubota Y. Curr. Opin. 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Untimely DNA synthesis could have catastrophic effects on cell viability or normal proliferative controls; therefore, prereplication complex assembly is permitted only during G1. The protein components of the prereplication complex are subject to a wide variety of controls, including phosphorylation, relocalization, and regulated protein accumulation through transcription and/or degradation, which restrict their activity to G1 and prevent the assembly of additional prereplication complexes on already replicated DNA. As a result, no part of the genome is replicated more than once per cell cycle (see Refs. 3Diffley J.F. Labib K. J. Cell Sci. 2002; 115: 869-872Crossref PubMed Google Scholar, 4Pelizon C. Trends Cell Biol. 2003; 13: 110-113Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 5Nishitani H. Lygerou Z. Genes Cells. 2002; 7: 523-534Crossref PubMed Scopus (223) Google Scholar, 6Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1397) Google Scholar, 7Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (334) Google Scholar, 8Blow J.J. EMBO J. 2001; 20: 3293-3297Crossref PubMed Scopus (71) Google Scholar). Studies in yeast and biochemical model systems have provided considerable insight into the function of prereplication complex proteins. However, it is apparent that the regulation of prereplication complex assembly in metazoans differs in important ways from that observed in yeasts or X. laevis egg extracts. For example, the subunits of the Mcm complex in Saccharomyces cerevisiae are translocated from the nucleus to the cytoplasm as S phase proceeds (17Labib K. Diffley J.F. Kearsey S.E. Nat. Cell Biol. 1999; 1: 415-422Crossref PubMed Scopus (171) Google Scholar, 18Yan H. Merchant A.M. Tye B.K. Genes Dev. 1993; 7: 2149-2160Crossref PubMed Scopus (190) Google Scholar), but mammalian Mcm proteins remain nuclear throughout the cell cycle and are released from chromatin into the nucleoplasm during S phase (19Kimura H. Nozaki N. Sugimoto K. EMBO J. 1994; 13: 4311-4320Crossref PubMed Scopus (147) Google Scholar, 20Todorov I.T. Pepperkok R. Philipova R.N. Kearsey S.E. Ansorge W. Werner D. J. Cell Sci. 1994; 107: 253-265Crossref PubMed Google Scholar, 21Musahl C. Schulte D. Burkhart R. Knippers R. Eur. J. Biochem. 1995; 230: 1096-1101Crossref PubMed Scopus (80) Google Scholar, 22Schulte D. Burkhart R. Musahl C. Hu B. Schlatterer C. Hameister H. Knippers R. J. Cell Sci. 1995; 108: 1381-1389Crossref PubMed Google Scholar). On the other hand, mammalian Cdc6 is relocalized from the nucleus to the cytoplasm in G2 (23Saha P. Chen J. Thome K.C. Lawlis S.J. Hou Z.H. Hendricks M. Parvin J.D. Dutta A. Mol. Cell. 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Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar), but recent reports indicate that metazoan Orc1 proteins are subject to regulated chromatin binding and ubiquitin-mediated degradation (30Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 31Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar, 32Tatsumi Y. Ohta S. Kimura H. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41528-41534Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Furthermore, the Cdt1 inhibitor protein, geminin, is apparently unique to metazoans, since no similar sequence has been identified in either yeast genome. Geminin binds to Cdt1 and is important for preventing inappropriate origin firing (13Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar, 33Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 34Mihaylov I.S. Kondo T. Jones L. Ryzhikov S. Tanaka J. Zheng J. Higa L.A. Minamino N. Cooley L. Zhang H. Mol. Cell. Biol. 2002; 22: 1868-1880Crossref PubMed Scopus (167) Google Scholar, 35Quinn L.M. Herr A. McGarry T.J. Richardson H. Genes Dev. 2001; 15: 2741-2754Crossref PubMed Scopus (146) Google Scholar, 36Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar). In addition, many of the genes that encode prereplication complex components are subject to transcriptional control in somatic cells, a level of regulation that does not apply to biochemical experimental systems. For these reasons, it is important to investigate the regulation of prereplication complex components in mammalian cells. Both Cdc6 and Cdt1 are required for the association Mcm complexes with chromatin, but it is not clear if they function as partners or if they function independently of one another. Cdc6 is an ATPase, and ATP binding and hydrolysis by Cdc6 is strictly required for DNA replication (37Weinreich M. Liang C. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 441-446Crossref PubMed Scopus (157) Google Scholar, 38Wang B. Feng L. Hu Y. Huang S.H. Reynolds C.P. Wu L. Jong A.Y. J. Biol. Chem. 1999; 274: 8291-8298Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 39Herbig U. Marlar C.A. Fanning E. Mol. Biol. Cell. 1999; 10: 2631-2645Crossref PubMed Scopus (65) Google Scholar). Conversely, the primary structure of Cdt1 does not suggest an enzymatic activity, and Cdt1 does not bear strong similarity to other proteins of known function. Cdt1 accumulates during G1 and S phase but is inhibited by association with the geminin protein, which accumulates during S phase and G2. Geminin binds to Cdt1 and inhibits the recruitment of Mcm proteins to chromatin, presumably by interfering with an essential Cdt1 function (13Tada S. Li A. Maiorano D. Mechali M. Blow J.J. Nat. Cell Biol. 2001; 3: 107-113Crossref PubMed Scopus (390) Google Scholar, 35Quinn L.M. Herr A. McGarry T.J. Richardson H. Genes Dev. 2001; 15: 2741-2754Crossref PubMed Scopus (146) Google Scholar, 36Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (584) Google Scholar, 40McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar). We sought to explore the molecular relationships between Cdc6, Cdt1, and the Mcm complex in a mammalian cell setting. In particular, we are interested in the processes by which replication complexes are assembled as cells leave a quiescent state and prepare for reentry into the cell cycle. In that regard, we have examined the protein-protein and protein-chromatin interactions involving Cdc6 and Cdt1 as quiescent cells are induced to grow. In this study, we provide evidence that Cdc6 and Cdt1 each require the function of the other in order to promote Mcm chromatin loading in mammalian cells. Cdc6 binds to Cdt1 via an essential N-terminal noncatalytic domain. Moreover, we find that the association of Cdt1 with Mcm2 (a subunit of the Mcm complex) is regulated by cell growth and that this interaction is influenced by Cdc6 and by geminin. These findings identify novel interactions between Cdt1, Cdc6, and geminin that contribute to the control of prereplication complex formation. Cells and Adenovirus—HeLa and 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. REF52 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum plus 5% calf serum and made quiescent by incubating in Dulbecco's modified Eagle's medium supplemented with 0.1% fetal calf serum plus 0.1% calf serum for 48 h. Restimulation was achieved by the addition of fetal calf serum to a final concentration of 20%. Cell cycle position was confirmed by staining with propidium iodide and flow cytometry (Cancer Center Flow Cytometry facility, Duke University Medical Center). Cells were infected with purified recombinant adenoviruses as previously described (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar, 42Nevins J.R. DeGregori J. Jakoi L. Leone G. Methods Enzymol. 1997; 283: 205-219Crossref PubMed Scopus (70) Google Scholar). Adenoviruses expressing green fluorescent protein and E2F3 and epitope-tagged Cdc6 have been described (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar, 43DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (610) Google Scholar). Adenovirus expressing native human Cdc6, epitope-tagged Cdc6 truncated after amino acid 192, Cdt1, and geminin were constructed by the method of He et al. (44He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar) and purified by CsCl banding as described (42Nevins J.R. DeGregori J. Jakoi L. Leone G. Methods Enzymol. 1997; 283: 205-219Crossref PubMed Scopus (70) Google Scholar). Fractionation of cells to obtain chromatin-enriched samples was performed as described (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar). Plasmid Constructions—Cdc6 was inserted into pAdTrack-CMV (44He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3256) Google Scholar) as a BamHI-NotI fragment excised from pBS-Cdc6 (gift of Zhen Yan). Cdt1 was excised as an EcoRI - NotI fragment from pGEX-Cdt1(gift of A. Dutta), the EcoRI site was made flush by treatment with Klenow, and the fragment was inserted into B-Shuttle-CMV (a derivative of pShuttle-CMV that lacks an irrelevant BamHI site) that had been digested with Asp718, made flush with T4 DNA polymerase, and then digested with NotI. A fragment containing the geminin open reading frame was produced from a cDNA-containing plasmid (gift of T. McGarry) in a PCR with oligonucleotide primers to generate a 5′ BamHI site and a 3′ EagI site flanking the open reading frame, digested with BamHI and EagI, and ligated to B-Shuttle-CMV that had been digested with BglII and NotI. The correct sequence of the resulting clone was confirmed. Adenovirus expressing amino acids 1-192 of Cdc6 was constructed by PCR amplification of the first 192 codons of Cdc6 with oligonucleotide primers that encode a BamHI site upstream of the initiator ATG at the 5′ end and a stop codon followed by a NotI site at the 3′ end. The resulting fragment was used to replace the full-length Cdc6 open reading frame in pJGC5, a shuttle vector that encodes five tandem copies of the c-Myc epitope tag at the N terminus of Cdc6 (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar), and the correct sequence was confirmed. N-terminal deletions of Cdc6 were constructed by PCR amplification of the 5′ portion of the Cdc6 cDNA in pcDNA3Myc5, and the resulting plasmids were sequenced. A plasmid to express hexahistidine-tagged geminin was constructed by inserting the geminin open reading frame as an EcoRI-HindIII fragment into the corresponding sites of pET28a (Novagen). A plasmid encoding the murine Cdc6 cDNA (pFastBac-mCdc6, a gift of F. Grummt (45Berger C. Strub A. Staib C. Lepke M. Zisimopoulou P. Hoehn K. Nanda I. Schmid M. Grummt F. Cytogenet. Cell Genet. 1999; 86: 307-316Crossref PubMed Scopus (8) Google Scholar)) was digested with NcoI, treated with Klenow enzyme, and then digested with NotI; the resulting fragment was ligated to pcDNA3 (Invitrogen) that had been digested with EcoRV and NotI. RNAi—A pool of double-stranded RNA specific to human Cdc6 (19-mers beginning with nucleotides 291, 587, 957, and 1278 of HSU77949) was obtained from Dharmacon (catalog no. M-003233-00-05). HeLa cells in 10-cm dishes were transfected with RNAi and plasmid at a final RNAi concentration of up to 0.75 μm using SuperFect reagent (Qiagen) for 40 h. In Vitro Protein Binding Assays—GST-Cdt1 was produced in BL21(DE3) cells from pGEX-Cdt1 by induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 2 h at 30 °C. GST was produced from the pGEX-5X-1 empty vector (Amersham Biosciences). Cells were pelleted and frozen at -80 °C. Frozen cells were resuspended in Buffer 6 (50 mm HEPES, pH 7.5, 0.1 m KAc, 0.02% Nonidet P-40, 10% glycerol, 5 mm MgCl2) containing protease inhibitors (0.1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 μg/ml pepstatin A, 10 μg/ml leupeptin, 1 μg/ml aprotinin) and 1 mg/ml lysozyme and sonicated three times for 30 s at moderate power 30% duty cycle. Triton X-100 was added to a final concentration of 1%, and lysates were incubated on ice for 30 min. After centrifugation at 12,000 × g, clarified supernatants were incubated with glutathione-Sepharose beads (Amersham Biosciences) at 4 °C for 2-3 h with constant rotation. Bound GST proteins were washed several times with cold Buffer 6 containing inhibitors and distributed to microcentrifuge tubes. REF52 cells (∼8 × 105 cells/sample) were harvested by trypsinization, washed once with 1× phosphate-buffered saline, and frozen as cell pellets at -80 °C. Cell pellets were thawed in 500 μl of Buffer 6 supplemented with the protease inhibitors listed above plus 10 μl of protease inhibitor mixture for mammalian cells (Sigma catalog no. P8340), 1 mm sodium orthovanadate, 25 mm β-glycerol phosphate, 5 μg/ml phosvitin, and 1 mm ATP. CaCl2 was added to a final concentration of 5 mm, and 15 units of micrococcal nuclease S7 (Roche Applied Science) were added to each cell suspension. Lysates were incubated on ice for 30 min and clarified by centrifugation at 13,000 × g for 2 min followed by centrifugation of the supernatant at 13,000 × g for 15 min at 4 °C. Portions of whole cell extracts were reserved on ice, and the remaining extracts were distributed to tubes containing bead-bound GST or GST-Cdt1. Equal amounts of total protein as determined by the Bio-Rad protein assay were added to each tube. Complexes were allowed to form at 4 °C with constant rotation for 2-3 h and then washed rapidly three times with 0.75 ml of ice-cold Buffer 6 with ATP, protease inhibitors, and phosphatase inhibitors. Proteins in whole cell extracts and proteins bound to beads were solubilized in 1× SDS sample buffer prior to SDS-PAGE and immunoblotting. Equal loading of both cellular proteins and GST proteins was confirmed by Ponceau S staining of the blots. Full-length human geminin was expressed as a hexahistidine-tagged fusion from the vector pET28a in BL21 cells as described for GST-Cdt1. Partial purification by nickel affinity chromatography was performed as directed by the manufacturer (Qiagen). Geminin eluted between 175 and 200 mm imidazole; protein concentration of geminin and full-length Cdt1 was estimated by Coomassie staining of dilutions of purified protein compared with known amounts of bovine serum albumin. Where indicated, geminin was added to GST-Cdt1 along with the cell extracts. As a control, an equivalent volume of the first elution fraction from the nickel column that contained no geminin detectable by Coomassie Blue staining was added. Co-immunoprecipitations were performed as above except that in place of the GST-Cdt1-coated beads, extracts were incubated with 10 μl of anti-Cdt1 antiserum or preimmune serum plus 10 μl of Protein A-agarose (Roche Applied Science). Cdc6 and geminin were produced from plasmids pcDNA3Myc5 or pET28a by transcription and translation in vitro using T7 polymerase and rabbit reticulocyte lysate in the presence of [35S]methionine according to the manufacturer's instructions (TNT Coupled Reticulocyte Lysate System; Promega). Proteins were separated from unincorporated methionine and small peptides by precipitation with 50% AmSO4 and resuspended in Buffer 6 prior to incubation with GST- or GST-Cdt1-coated beads as described above. Antibodies—Anti-Cdt1 antiserum was raised in guinea pigs using recombinant GST-Cdt1 as antigen (Cocalico Biologicals). Anti-mouse Cdt1 was a gift from F. Hanaoka. Anti-Mcm2 was obtained from Transduction Laboratories (catalog no. B58720). Anti-Cdc6 (sc-9964), anti-geminin (sc-13015), anti-c-Myc epitope tag (sc-40), and anti-actin (sc-8432) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse and anti-rabbit secondary antibodies coupled to horseradish peroxidase were obtained from Amersham Biosciences; anti-guinea pig coupled to horseradish peroxidase was obtained from Jackson ImmunoResearch Laboratories. Cdc6 and Cdt1 Are Each Required for Mcm Chromatin Loading in Vivo—Deletion of the CDC6 gene from yeast cells prevents the normal association of Mcm proteins with chromatin during G1 (10Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 16Donovan S. Harwood J. Drury L.S. Diffley J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (433) Google Scholar, 46Cocker J.H. Piatti S. Santocanale C. Nasmyth K. Diffley J.F. Nature. 1996; 379: 180-182Crossref PubMed Scopus (296) Google Scholar, 47Perkins G. Diffley J.F. Mol. Cell. 1998; 2: 23-32Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Interfering with Cdc6 function in mammalian cells by expression of mutationally altered forms or microinjection of anti-Cdc6 antibodies blocks S phase entry (39Herbig U. Marlar C.A. Fanning E. Mol. Biol. Cell. 1999; 10: 2631-2645Crossref PubMed Scopus (65) Google Scholar, 48Yan Z. DeGregori J. Shohet R. Leone G. Stillman B. Nevins J.R. Williams R.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3603-3608Crossref PubMed Scopus (214) Google Scholar). Presumably, these latter results are the consequence of failure of Cdc6 to load Mcm complexes onto chromatin, as is seen in model systems. We have previously shown that human Cdc6 has the ability to stimulate Mcm chromatin loading in mammalian cells (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar); we have now directly explored the role of Cdc6 in the loading of Mcm proteins in mammalian cells using RNAi to achieve loss of Cdc6. We transfected growing human cells with a pool of double-stranded RNA specific to human Cdc6 (RNAi). Increasing amounts of RNAi led to a substantial loss of endogenous Cdc6 protein, as measured by immunoblotting of whole cell extracts with anti-Cdc6 antibody (Fig. 1A). We then examined the chromatin-bound proteins in cells lacking Cdc6 by extracting the soluble proteins with detergent and isolating the insoluble fraction, as previously described (41Cook J.G. Park C.H. Burke T.W. Leone G. DeGregori J. Engel A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1347-1352Crossref PubMed Scopus (101) Google Scholar). We tracked Mcm complexes in whole cell extracts (Fig. 1B, lanes 1-3) and bound to chromatin (Fig. 1B, lanes 4-6) by probing immunoblots for one of the six Mcm subunits, Mcm2. Actin, which is also contained in the detergent-insoluble fraction (although not bound to chromatin), was monitored as a loading control. When endogenous Cdc6 levels are reduced, Mcm2 is poorly associated with chromatin (Fig. 1B, compare lanes 4 and 5). In order to demonstrate that the effects on Mcm2 chromatin loading are specific to the loss of Cdc6, we co-transfected cells with both the Cdc6 RNAi and a plasmid for expression of mouse Cdc6. The sequences targeted by the Cdc6 RNAi have a minimum of three mismatches each with the murine Cdc6 cDNA. When Cdc6 production is maintained by the mouse clone (Fig. 1B, lane 3), Mcm2 chromatin association is similarly maintained (Fig. 1B, lane 6), indicating that Cdc6 is specifically required for the normal recruitment of the Mcm complex to chromatin in intact mammalian cells. Like Cdc6, deletion of the yeast CDT1 gene blocks the recruitment of Mcm complexes to chromatin (11Devault A. Vallen E.A. Yuan T. Green S. Bensimon A. Schwob E. Curr. 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We wished to determine whether the activity of Cdc6 under these conditions required Cdt1 activity or if ectopic expression of Cdc6 independently induced the loading of Mcm proteins onto chromatin without a contribution from Cdt1. In or