The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase Joon-Kyu Lee*†, Yeon-Soo Seo†, and Jerard Hurwitz*‡ *Program in Molecular Biology, Memorial Sloan–Kettering Cancer Center, New York, NY 10021; and †Department of Biological Science, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea Contributed by Jerard Hurwitz, December 4, 2002 Previous studies in Saccharomyces cerevisiae have defined an (9). Furthermore, studies in S. cerevisiae with mcm degron essential role for the Dbf4-Cdc7 kinase complex in the initiation of mutants showed that the Mcm proteins are also required for the DNA replication presumably by phosphorylation of target proteins, progression of the replication fork (10). These observations such as the minichromosome maintenance (Mcm) complex. We suggest that the Mcm complex is the replicative helicase. have examined the phosphorylation of the Mcm complex by the Cdc7, a serine͞threonine kinase conserved from yeast to Dfp1-Hsk1 kinase, the Schizosaccharomyces pombe homologue of humans (reviewed in ref. 11), is activated by the regulatory Dbf4-Cdc7. In vitro, the purified Dfp1-Hsk1 kinase efficiently phos- protein Dbf4. Although the level of Cdc7p is constant through- ͞ phorylated Mcm2p. In contrast, Mcm2p, present in the six-subunit out the cell cycle, the activity of this kinase peaks at the G1 S Mcm complex, was a poor substrate of this kinase and required transition, concomitant with the cellular level of Dbf4p. In S. ͞ Cdc23p (homologue of Mcm10p) for efficient phosphorylation. In cerevisiae, Dbf4p binds to chromatin at the G1 S transition and the presence of Cdc23p, Dfp1-Hsk1 phosphorylated the Mcm2p and remains attached to chromatin during S phase (12). In the Mcm4p subunits of the Mcm complex. Cdc23p interacted with both Xenopus cell free DNA replication system, Cdc7p was found to the Mcm complex and Dfp1-Hsk1 by selectively binding to the bind to chromatin during the G1 and S phase and this association Mcm4͞6͞7 subunits and Dfp1p, respectively. The N terminus of required the Mcm complex (13). Mcm proteins interact with Cdc23p was found to interact directly with Dfp1-Hsk1 and was Dbf4p-Cdc7p and Mcm2p is a good substrate of the kinase in vivo essential for phosphorylation of the Mcm complex. Truncated and in vitro (12, 14). Cells containing an allele of MCM5, derivatives of Cdc23p that complemented the temperature-sensi- mcm5-bob1, bypass the requirements for CDC7 and DBF4 (15), tive phenotype of cdc23 mutant cells also stimulated the phos- suggesting that the Mcm complex is the major target of the Cdc7 phorylation of Mcm complex, implying that this activity might be kinase. a critical role of Cdc23p in vivo. These results suggest that Cdc23p MCM10 was initially identified in S. cerevisiae as a gene participates in the activation of prereplicative complex by recruit- required for chromosomal DNA replication and stable plasmid ing the Dfp1-Hsk1 kinase and stimulating the phosphorylation of maintenance (16, 17). Homologues of this gene have been the Mcm complex. identified in other organisms, including S. pombe, Xenopus, and human (18–20). Studies in S. cerevisiae showed that MCM10 is DNA replication ͉ scaffold protein essential for the initiation of replication and interacts genetically with many genes involved in the initiation and elongation steps of DNA replication. These included MCM, CDC45, ORC, DNA2, he initiation of eukaryotic DNA replication is a multistep ␧ ␦ process that commences with the binding of the origin and genes encoding DNA polymerase and . Physical inter- T action of Mcm10p with the Mcm complex and Orc proteins have recognition complex (Orc) to origins. During the G1 phase of the cell cycle, components of the prereplicative complex (pre-RC), also been demonstrated (16, 18). In S. cerevisiae, Mcm10p was such as Cdc6͞Cdc18, Cdt1, and the minichromosome mainte- reported to be a component of the pre-RC and required for the nance (Mcm) complex, are recruited sequentially to origin DNA association of the Mcm complex with origin DNA. In the in an Orc-dependent manner. Upon entering S phase, replication Xenopus replication system, the binding of Mcm10p onto chro- is initiated by the activation of pre-RC, which leads to the matin required the presence of chromatin-bound Mcm complex binding of Cdc45, subsequent unwinding of replication origins, and Mcm10p was required for loading Cdc45p and origin and the recruitment of the replication fork machinery. Two S unwinding (20). phase-promoting kinases, the S-phase cyclin-dependent protein Although the six-subunit Mcm complex appears to be a major kinase (Cdk) and Dbf4-Cdc7 kinase, are essential for the acti- target of the Cdc7 kinase, there is little information available vation of the pre-RC in budding yeast. Although in vivo targets about the phosphorylation of the Mcm complex by this kinase. of the S-phase Cdk are unclear, the Mcm complex appears to be To investigate the role of the Cdc7 kinase and the biological a major target of the Cdc7 kinase (1–4). consequences of its phosphorylation of the Mcm complex, we S. pombe The Mcm complex is composed of six subunits (Mcm2, Mcm3, have reconstituted the Dfp1-Hsk1 kinase complex, the homologue of Dbf4-Cdc7 kinase, and examined its phosphory- Mcm4, Mcm5, Mcm6, and Mcm7), which are all structurally lation of the Mcm complex in vitro. Although the purified related and highly conserved in eukaryotes (5, 6). All six proteins Dfp1-Hsk1 kinase phosphorylated Mcm2p efficiently, phosphor- are essential for the assembly of the pre-RC and DNA replica- ylation of the six-subunit Mcm complex was less effective. We tion. Although the role of this complex in DNA replication is not identified Cdc23p as an additional factor required for the fully understood, in vivo and in vitro studies suggest that the Mcm efficient phosphorylation of the Mcm complex. We present data proteins may play a role as a replicative helicase (1). Biochemical suggesting that Cdc23p may play an important role in the analysis of the Mcm complex showed that the human and Schizosaccharomyces pombe Mcm4͞6͞7 complex contains DNA helicase activity in vitro (7, 8). In vivo cross-linking and chromatin Abbreviations: HA, hemagglutinin; Mcm, minichromosome maintenance; Orc, origin rec- immunoprecipitation experiments performed in Saccharomyces ognition complex; pre-RC, prereplicative complex; ts, temperature sensitive; IVT, in vitro cerevisiae showed that the localization of the Mcm proteins transcription and translation. shifted from origin regions to inter-origin regions during S phase ‡To whom correspondence should be addressed. E-mail: [email protected]. 2334–2339 ͉ PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0237384100 Downloaded by guest on October 1, 2021 activation of the pre-RC by recruiting Dfp1-Hsk1 kinase into the H containing 0.2 M sodium glutamate. Proteins bound to the pre-RC through its interaction with the regulatory subunit Dfp1, beads were analyzed by SDS͞9% PAGE followed by Western leading to the stimulation of the phosphorylation of Mcm blot analysis or autoradiography [for in vitro transcribed and complex by this kinase. translated (IVT) proteins]. Materials and Methods Complementation of cdc23 Temperature-Sensitive (ts) Phenotype by Purification of Dfp1-Hsk1 Kinase Complex, Cdc23p, and Mcm Complex. Truncated Cdc23 Proteins. To examine the complementation of the Baculoviruses expressing the S. pombe Dfp1 or Hsk1 protein cdc23 ts phenotype (18), cDNA fragments encoding truncated were prepared from cDNAs encoding full-length proteins that Cdc23 proteins were subcloned into the S. pombe expression were subcloned into the plasmid pFastBac1 (Life Technologies, vector pREP1 containing the nmt1 promoter. After transfor- Rockville, MD). Two FLAG and three hemagglutinin (HA) mation of these plasmids into the S. pombe strain, hϪ ade6-M216 epitope tags were added at the N terminus of Dfp1 and C leu1–32 ura4-D18 his3-D1 cdc23-M36, complementation of the ts terminus of Hsk1 proteins, respectively, to facilitate their de- phenotype was examined by cell growth in minimal medium tection and purification. For the purification of Dfp1-Hsk1 plates lacking thiamine (induced condition) at 36.5°C for 4 days. kinase complex, Sf9 cells (2 ϫ 106 cells per ml, 300 ml) were infected with baculoviruses expressing these two proteins and Results incubated at 27°C for 60 h. Cells were harvested, washed with Purification and Biochemical Properties of the Dfp1-Hsk1 Kinase ice-cold PBS, and resuspended with 20 ml of buffer H (25 mM Complex. To examine the biochemical properties of Dfp1-Hsk1 ͞ ͞ ͞ ͞ Hepes-NaOH, pH 7.5 5 mM MgCl2 1 mM EGTA 1mMDTT kinase, the complex was reconstituted by using the baculovirus 0.05% Nonidet P-40͞10% glycerol) containing 0.15 M sodium expression system and purified. This procedure yielded the glutamate, 50 mM ␤-glycerophosphate, 15 mM p-nitrophenyl Dfp1-Hsk1 complex that was Ϸ90% pure (Fig. 1A), which was phosphate, 0.1 mM sodium vanadate, 1 mM PMSF, 1 mM free of contamination by other kinases such as cyclin-dependent benzamidine⅐HCl, 10 ␮g͞ml leupeptin, 10 ␮g͞ml pepstatin A, 5 protein kinase. Western blot analysis showed that both regula- ␮g͞ml aprotinin, and 0.5% Triton X-100. After incubation on ice tory and catalytic subunits migrated to the same position in for 20 min, the supernatant solution was collected by centrifu- SDS͞PAGE (data not presented), as reported (14). This kinase gation at 35,000 ϫ g at 4°C for 30 min, mixed with 1 ml of preparation phosphorylated Mcm2p at a maximum rate of about anti-FLAG M2 Ab agarose (Sigma) beads, and incubated at 4°C 1.5 pmol͞min per pmol of kinase (Fig. 1 B and C). It should be each time for 3 h with rocking. The beads were collected, washed noted that the phosphorylated form of Mcm2p migrated faster BIOCHEMISTRY with 15 ml of buffer H four times, and eluted three times by than unphosphorylated Mcm2p on SDS͞PAGE (see Fig.