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• W2061065780 abstract "The yeast Saccharomyces cerevisiaeCdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1∼5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of variousdbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex. The yeast Saccharomyces cerevisiaeCdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1∼5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of variousdbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex. autonomously replicating sequence hemagglutinin polyacrylamide gel electrophoresis glutathione S-transferase alkaline phosphatase hydroxyurea Initiation of chromosomal DNA replication and cell cycle progression are tightly regulated in eukaryotes. In the yeastSaccharomyces cerevisiae, several cdc(cell division cycle) mutants that block initiation of chromosomal DNA replication have been isolated and characterized (1Hartwell L.H. J. Bacteriol. 1974; 115: 966-974Crossref Google Scholar, 2Hartwell L.H. J. Mol. Biol. 1976; 104: 803-817Crossref PubMed Scopus (193) Google Scholar), for example, cdc28, cdc4,cdc6, and cdc7. The stepwise assembly of proteins at origins of DNA replication is a crucial part of regulating entry into S phase. Two key factors that mediate such cell cycle regulation are Cdc6 protein level and availability and the presence of an active cyclin-dependent kinase (Cdk) (see Ref. 3Stillman B. Science. 1996; 274: 1659-1663Crossref PubMed Scopus (428) Google Scholar for review). The origin recognition complex is bound to origins of DNA replication at all stages of the cell cycle in S. cerevisiae (4Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 5Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar, 6Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). However, Cdc6 is not recruited to origins until late in M phase and is required for the association of the Mcm2–7 family proteins at origins to form a prereplicative complex (6Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 7Detweiler C.S. Li J.J. J. Cell Sci. 1997; 110: 753-763PubMed Google Scholar, 8Donovan S. Harwood J. Drury L.S. Diffley J.F.X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (428) Google Scholar). The Cdc6 protein-dependent stage of the assembly reaction is inhibited by active Clb-Cdks (6Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 9Dahmann C. Diffley J.F.X. Nasmyth K.A. Curr. Biol. 1995; 5: 1257-1269Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 10Piatt S. Bohm T. Cocker J.H. Diffley J.F.X. Nasmyth K. Genes Dev. 1996; 10: 1516-1531Crossref PubMed Scopus (253) Google Scholar). Because Cdc6 protein is synthesized only from late M phase until late G1 (11Piatt S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (333) Google Scholar), prereplicative complexes can only be assembled during this period of the cell cycle. S phase cyclin-Cdk (Cdc28p/Clb5p or Cdc28p/Clb6p) activity is required for the chromatin association of Cdc45p just before the initiation of chromosomal DNA replication (12Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (273) Google Scholar). The previous results demonstrated that Cdc28 protein-Clb kinase is required throughout S phase to activate origins when they are scheduled to fire (13Donaldson A.D. Raghuraman M.K. Friedman K.L. Cross F.R. Brewer B.J. Fangman W.L. Mol. Cell. 1998; 2: 173-182Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The Cdc7/Dbf4 complex is a Cdk-like protein kinase (see Refs. 14Johnston L.H. Masai H. Sugino A. Trends Cell Biol. 1999; 9: 249-252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar and 15Oshiro G. Owens J.C. Shellman Y. Sclafani R.A. Li J.J. Mol. Cell. Biol. 1999; 19: 4888-4896Crossref PubMed Scopus (99) Google Scholarfor review) that is also required for entry into S phase at a very late stage. CDC7 transcript levels are constant throughout the cell cycle, whereas DBF4 transcription is cell cycle regulated and the transcript abundance peaks near the G1 to S phase transition. The DBF4 gene was first identified as a gene that could suppress cdc7 mutants when present in multiple copies (16Kitada K. Johnston L.H. Sugino T. Sugino A. Genetics. 1992; 131: 21-29Crossref PubMed Google Scholar), and Dbf4p is now known to be required for Cdc7p-mediated kinase activity. It has been shown that a Cdc7p-associated H1 kinase activity is cell cycle regulated (17Jackson A.L. Pahl P.M.B. Harrison K. Rosamond J. Sclafani R.A. Mol. Cell. Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar). It is not clear, however, whether this H1 kinase activity reflects the normal activity of the Cdc7p kinase. Recently, homologues of Cdc7p have been identified in Schizosaccharomyces pombe (18Masai H. Miyake T. Arai K. EMBO J. 1995; 14: 3094-3104Crossref PubMed Scopus (124) Google Scholar), mouse (19Kim J.M. Sato N. Yamada M. Arai K. Masai H. J. Biol. Chem. 1998; 273: 23248-23257Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar),Xenopus, and humans (20Jiang W. Hunter T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14320-14325Crossref PubMed Scopus (67) Google Scholar, 21Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar, 22Hess G.F. Drong R.F. Weiland K.L. Slightom J.L. Sclafani R.A. Hollingsworth R.E. Gene (Amst. ). 1998; 211: 133-140Crossref PubMed Scopus (57) Google Scholar), and a Dbf4p homologue has also been found in S. pombe (23Brown G.W. Kelly T.J. J. Biol. Chem. 1998; 273: 22083-22090Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 24Takeda T. Ogino K. Matsui E. Cho M.K. Kumagai H. Miyake T. Arai K. Masai H. Mol. Cell. Biol. 1999; 19: 5535-5547Crossref PubMed Scopus (93) Google Scholar) and human (25Kumagai H. Sato N. Yamada M. Mahony D. Seghezzi W. Lees E. Arai K. Masai H. Mol. Cell. Biol. 1999; 19: 5083-5095Crossref PubMed Scopus (110) Google Scholar). The presence of these homologues in evolutionarily distant species suggests that Cdc7p/Dbf4p plays a conserved role in the initiation of DNA replication in eukaryotes. Several lines of evidence suggest that Cdc7p/Dbf4p acts directly at origins of DNA replication and that the Mcm proteins may be one of the targets of the kinase. DBF4 was isolated in a one-hybrid screen for autonomously replicating sequence (ARS)1-interacting factors (26Dowell S.J. Romanoski P. Diffley J.F.X. Science. 1994; 265: 1243-1246Crossref PubMed Scopus (172) Google Scholar). Interestingly, its interaction with origins was found to be independent of its ability to bind to Cdc7p. Two recent reports (27Bousset K. Diffley J.F.X. Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (238) Google Scholar,28Donaldson A.D. Fangman W.L. Brewer B.J. Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar) suggest that Cdc7p activity is required to activate individual origins rather than for a global activation of S phase, because temperature-sensitive cdc7 mutants demonstrate an inability to activate late rather than early replication origins at a semi-permissive temperature. Potential target(s) of Cdc7p that are required for the initiation of DNA replication were revealed through genetic analysis of a mutant in S. cerevisiae that could bypass the requirement for CDC7 or DBF4 (17Jackson A.L. Pahl P.M.B. Harrison K. Rosamond J. Sclafani R.A. Mol. Cell. Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar). The mutation was found to reside in MCM5/CDC46 (29Hardy C.F.J., O. Dryga O. Pahl P.M.B. Sclafani R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3151-3155Crossref PubMed Scopus (217) Google Scholar), a member of the Mcm family of six sequence-related proteins (30Tye B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2399-2401Crossref PubMed Scopus (54) Google Scholar). Since then, a number of reports have suggested that some Mcm proteins such as Mcm2 and Mcm3 are in vitro substrates of Cdc7p kinases from various sources (21Sato N. Arai K. Masai H. EMBO J. 1997; 16: 4340-4351Crossref PubMed Scopus (121) Google Scholar, 23Brown G.W. Kelly T.J. J. Biol. Chem. 1998; 273: 22083-22090Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar). Another study recently reported that all yeast Mcm proteins, except for Mcm5 expressed and prepared from insect cells, were substrates for the Cdc7/Dbf4p protein kinase complex (32Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Crossref PubMed Scopus (227) Google Scholar). RAD53 encodes a dual specificity protein kinase that is required for all three DNA damage checkpoints at G1, S phase, and G2 (33Sanchez Y. Desany B.A. Jones W.J. Liu Q. Wang B. Elledge S.J. Science. 1996; 271: 357-360Crossref PubMed Scopus (527) Google Scholar). It is thought to be part of the transducer class (34Weinert T. Curr. Opin. Genet. Dev. 1998; 8: 185-193Crossref PubMed Scopus (174) Google Scholar). Several lines of circumstantial evidence suggest that in addition to its checkpoint function, Rad53p could be also involved in the regulation of temporal order origin firing during chromosomal DNA replication (35Santocanale C. Diffley J.F.X. Nature. 1998; 395: 615-618Crossref PubMed Scopus (525) Google Scholar). In this report, we describe the expression and the purification of recombinant yeast Cdc7p/Dbf4p to near homogeneity and biochemical analysis of the Cdc7p/Dbf4p complex-associated protein kinase activity. We show that both subunits of the recombinant enzyme are auto-phosphorylated and that this reaction requires active Dbf4p and Cdc7p. The recombinant protein efficiently phosphorylates Mcm2 and also phosphorylates with somewhat lower efficiency the remaining Mcm proteins as well as the largest subunit of DNA polymerase α-primase and RPA. However, when Mcm2 protein was pretreated with an alkaline phosphatase, it was not phosphorylated by the Cdc7p/Dbf4p kinase, suggesting that Mcm2 protein must be prephosphorylated by an unknown kinase(s) to be a substrate for Cdc7/Dbf4 protein kinase. Analysis of mutant Cdc7/Dbf4 protein kinases suggests that ARS-binding and Cdc7 protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex that functions in chromosomal DNA replication. Finally, inhibition of the Cdc7p/Dbf4p complex kinase activity by Rad53p-mediated phosphorylation is demonstrated. Based on these results, it is proposed that Rad53p regulates the temporal activation of chromosomal DNA replication origins in yeast by regulating the activity of Cdc7p/Dbf4p protein kinase. The following yeast strains were used in this study: S. cerevisiae 208 (MATa cdc7-1 ura3-52 leu2-3, 112), L128-2D (MATa dbf4-1 ura3 trp1), L202-1A (MATa dbf4-2 ura3 leu2 trp1), KKY743 (MATα dbf4-3 ura3 leu2 trp1), KKY744 (MATα dbf4-4 ura3 leu2 trp1), and KKY745 (MATα dbf4-5 ura3 leu2 trp1) were previously described (16Kitada K. Johnston L.H. Sugino T. Sugino A. Genetics. 1992; 131: 21-29Crossref PubMed Google Scholar). YKY9 (MATa leu2 trp1-298 ura3-52 prb ΔPEP4::TRP1), and YKY30 (MATαura3 leu2 trp1 bob1 ΔCDC7::LEU2 ΔPEP4::TRP1) were used for expression and purification of GST-Mcm 2–7 proteins. CB001–2HA-Rad53 (MATα leu2 trp1 ura3 prb pep4::URA3 RAD53::[YCpRAD53-HA]) was constructed by transforming S. cerevisiae CB001 (36Hamatake R.K. Hasegawa H. Clark A.B. Bebenek K. Kunkel T.A. Sugino A. J. Biol. Chem. 1990; 265: 4072-4083Abstract Full Text PDF PubMed Google Scholar) with 2HA-taggedRAD53 plasmid (Ycp-RAD53-HA) (37Sugimoto K. Ando S. Shimomura T. Matsumoto K. Mol. Cell. Biol. 1997; 17: 5905-5914Crossref PubMed Scopus (84) Google Scholar) digested with restriction enzyme BamHI. The dbf4-1 sup1 was isolated from spontaneous revertants of temperature-sensitive L128-2D strain. Standard DNA manipulations were carried out according to Sambrook et al. (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Oligonucleotides 5′-GGCCAGATCTCATAATGACAAGCAAAACGA-3′, 5′-GGCCAGATCTTTGCTATTCAGATATTAGG-3′, 5′-GGCCGGATCCAAGAAAATGGTTTCTCCAACGAAA-3′, and 5′-GGCCGGATCC CTATATTTGAAATCTGAGATT-3′ (underlined nucleotides represent either BglII orBamHI sites, and bold nucleotides are the initiation or termination codons) were synthesized on an automated DNA synthesizer (Beckman) and were used for polymerase chain reaction amplification of the DNA fragments containing CDC7 and DBF4 on the plasmid pKK709 and pKK616 DNA (16Kitada K. Johnston L.H. Sugino T. Sugino A. Genetics. 1992; 131: 21-29Crossref PubMed Google Scholar). Amplified DNA was digested with either BglII (for CDC7) or BamHI enzyme (for DBF4) and inserted into either BglII- or BamHI-digested baculovirus vector pBacPAK9. The resulting constructs, pBac-Cdc7p and pBac-Dbf4p, were co-transfected withBsu36I-linealized wild type virus BacPAK6 viral DNA into Sf9 cells using cationic liposome-mediated transfection according to the Invitrogen protocol, and recombinant virus was plaque purified. The presence of an insert in a putative recombinant virus was verified by polymerase chain reaction using primers complementary to the polyhedron locus. Recombinant virus Bac-Cdc7p 17 and Bac-Dbf4p 32 were amplified in Sf9 cells to a titer of 108plaque-forming units/ml. Recombinant virus containing mutantcdc7-1 or dbf4-1∼5 were constructed as wild type CDC7 and DBF4 genes. Sf9 cells were grown to confluence in T-75 Falcon flasks at 27 °C in Grace's medium supplemented with 10% fetal bovine serum, 50 μg/ml gentamycin, and 0.1% pleuronic F-68 and infected with recombinant virus pBac-Cdc7 17 or pBac-Dbf4 32 at a multiplicity of infection of 10. Cells were harvested at 72 h post-infection by centrifugation at 1,200 × g and washed twice with serum-free Grace's medium. Recombinant virus pBac-Cdc7 17 and pBac-Dbf4 32 were used to infect 2 L of Sf9 cells (2 × 106cells/ml) at a multiplicity of infection of 10. After 72 h the cells were harvested, washed with serum-free Grace's medium, resuspended in 60 ml of ice-cold HSL buffer (50 mmTris-HCl, pH 7.4, 1% (v/v) Nonidet P-40, and 500 mm NaCl) containing a mixture of protease inhibitors (10 μg/ml aprotinin, 5 μg/ml leupeptin, 100 μg/ml bacitracin, 250 μg/ml soybean trypsin inhibitor, 1 mm phenylmethylsulfonyl fluoride, and 10 mm benzamidine), and subjected to sonication by Ultrosonic Disruptor UD-201 (Tomy, Japan). To the cell extract (52 ml), obtained by centrifugation at 27,000 × g for 20 min, 16.3 g of solid (NH4)2SO4 (50% saturation) was gradually added with stirring. After 30 min stirring, the extract was centrifuged at 27,000 × g for 20 min. The precipitated protein was dissolved in 30 ml of buffer A (50 mm Tris-HCl, pH 7.5, 10 mm 2-mercaptoethanol, 1 mm EDTA, 10% glycerol (v/v), and 1 mmphenylmethylsulfonyl fluoride) and dialyzed against 2 liters of buffer A containing 50 mm NaCl for 4 h at 0 °C. After centrifugation at 27,000 × g for 20 min, the suspension was loaded onto an S-Sepharose column (80 ml) equilibrated in buffer A containing 100 mm NaCl. The column was washed with three bed volumes of the same buffer, and the protein was eluted with a 320-ml linear gradient of 100–600 mm NaCl in buffer A. Fractions containing Cdc7p and Dbf4p, as identified by activity assays and Western blot analysis (0.2–0.3 m NaCl), were pooled and loaded onto a Hi Trap Heparin column (5 ml) equilibrated with buffer A containing 200 mm NaCl. After washing with three bed volumes of equilibration buffer, protein was eluted with a 120-ml linear gradient of 0.2–1 m NaCl in buffer A. The peak fractions (0.5–0.65 m NaCl) were pooled, dialyzed twice against 1 liter of buffer A containing 100 mm NaCl for 3 h, and loaded onto a Mono Q column (HR5/5, 1 ml) pre-equilibrated with buffer A containing 100 mm NaCl. Protein was eluted with a 15-ml linear gradient from 100 to 700 mm NaCl in buffer A. Active fractions (0.3–0.4m NaCl) were pooled together, dialyzed against 50% glycerol, 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 10 mm 2-mercaptoethanol, and 1 mmphenylmethylsulfonyl fluoride for 2 h and stored at −80 °C. S. cerevisiae CB001 cells expressing 2HA-tagged Rad53p were grown at 30 °C in 24L YPD medium toA 600 = 1–2 and harvested by centrifugation at 8,000 rpm for 20 min. Cells were resuspended in buffer A (36Hamatake R.K. Hasegawa H. Clark A.B. Bebenek K. Kunkel T.A. Sugino A. J. Biol. Chem. 1990; 265: 4072-4083Abstract Full Text PDF PubMed Google Scholar) to 1 g/ml and disrupted by passage through a Gaulin homogenizer at 10,000 psi. To cell extracts, NaCl was added to a final concentration of 0.5 m. After stirring at 0 °C for 30 min, cell debris was removed by centrifugation in a Beckman JLA10.5 rotor at 8,000 rpm for 20 min, and the supernatant was saved. To the supernatant, 10%(w/v) Polymin P (pH 8) solution was slowly added to 0.5% (final concentration), mixed for 15 min, and centrifuged in a Beckman JLA 10.5 rotor at 8000 rpm for 20 min. 0.313 g of ammonium sulfate was added per ml of supernatant, and the mixture was stirred for 30 min. The protein precipitate was collected by centrifugation in a Beckman JLA 10.5 rotor at 10,000 rpm for 30 min. The pellet was stored at −80 °C. The precipitate was resuspended in 20 ml of buffer A and dialyzed against 2 liters of buffer A for 2 h and 2 liters of 0.1 m NaCl in buffer A for 2 h. Insoluble material was removed from the dialysate by centrifugation in a Beckman JA20.0 rotor at 10,000 rpm for 10 min. The supernatant was applied on an S-Sepharose column (75 ml) equilibrated with 0.1 m NaCl in buffer A, the column was washed with three column volumes of 0.1m NaCl in buffer A, and protein was eluted with one column volume of 0.5 m NaCl in buffer A. The protein was eluted at 0.5 m NaCl. Fractions were checked by Western blot analysis followed by immunostaining. The fractions containing the protein was pooled together (Fraction I). Fraction I was dialyzed twice against 2 liters of 0.05 m NaCl in buffer A for 2 h and centrifuged in a Beckman JA20.0 rotor at 10,000 rpm for 10 min to remove any precipitates. The supernatant was applied on a Mono Q column (HR 10/10) equilibrated with 0.05 m NaCl in buffer A. The column was washed with three column volumes of 0.05 m NaCl in buffer A, and then protein was eluted with 20 column volumes of linear gradient from 0.05 to 0.5m NaCl in buffer A. Fractions containing the Rad53 protein were identified by Western blot analysis and pooled (Fraction II). Fraction II was dialyzed twice against 2 liters of 0.1 m NaCl in buffer A for 2 h and applied on a Mono S column (HR 5/5) equilibrated with 0.1 mNaCl in buffer A. The column was washed with three column volumes of 0.1 m NaCl in buffer A and then eluted with a 30-ml linear gradient of 0.1–0.5 m NaCl in buffer A. Fractions containing Rad53p were identified by protein kinase activity assay using Histone H1 and Western blot analysis, and the active fractions were pooled (Fraction III). Fraction III was dialyzed against 1 liter of 0.1 m NaCl in buffer A for 3 h and applied on a Hi-Trap Heparin column (HR5/5) equilibrated with 0.1 m NaCl in buffer A. The column was washed with three column volumes of 0.1m NaCl in buffer A and then eluted with 20 ml of linear gradient of 0.1–0.5 m NaCl in buffer A. Fractions containing Rad53 protein were identified by protein kinase activity assays and Western blot analysis, and the active fractions were pooled (Fraction IV). HU-activated Rad53p was also purified as described above from CB001 cells expressing 2HA-tagged Rad53p after treating cells with 0.2m hydroxyurea for 3 h at 30 °C. More detailed characterization of the purified 2HA-tagged Rad53p will be published elsewhere. 4–20% gradient SDS-PAGE was performed by the method of Laemmli (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Protein concentration was determined by the method of Bradford with bovine serum albumin as standard as described previously (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar, 36Hamatake R.K. Hasegawa H. Clark A.B. Bebenek K. Kunkel T.A. Sugino A. J. Biol. Chem. 1990; 265: 4072-4083Abstract Full Text PDF PubMed Google Scholar). Following SDS-PAGE, proteins were electroblotted onto an Immobilon-P (Millipore) membrane that was incubated for 30 min with blocking buffer, Tris-buffered saline (50 mm Tris-HCl, pH 7.4, and 100 mm NaCl) containing 2% nonfat dry milk, and incubated overnight with rabbit polyclonal antipeptide antiserum to Dbf4p (provided by J. Diffley) or with rabbit polyclonal antiserum to Cdc7p expressed inEscherichia coli (provided by H. Masai). After washing three times with blocking buffer, the membrane was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG for 1.5 h at room temperature, washed three times with blocking buffer, and washed twice with Tris-buffered saline. Color was developed as described previously (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar). Protein kinase activity was assayed with GST-Mcm2p purified from S. cerevisiae as a substrate. Reaction mixtures (20 μl) contained 20 mm Hepes, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 10% (v/v) glycerol, 300 pmol of [γ-32P]ATP (specific activity, 10,000 cpm/pmol), 10–30 pmol of GST-Mcm2p, and Cdc7p/Dbf4p protein kinase complex or cell extracts. After 5 min at 30 °C, reactions were stopped by the addition of 5 μl of stop solution, heated at 95 °C for 3 min, and subjected to 4–20% SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Brilliant Blue, destained, dried and autoradiographed (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar). To measure radioactivity incorporated into the protein, the corresponding protein bands were excised from the gel and quantitated in the presence of scintillation fluid using a scintillation spectrometer. The purification of yeast GST-Mcm2-Mcm7 from S. cerevisiae was described previously (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar), and their purity was more than 95% (data not shown). Calf thymus histone H1 (more than 95% pure) was obtained from Nacalai Tesque, Japan. Other methods and materials used in this study were previously described (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar, 36Hamatake R.K. Hasegawa H. Clark A.B. Bebenek K. Kunkel T.A. Sugino A. J. Biol. Chem. 1990; 265: 4072-4083Abstract Full Text PDF PubMed Google Scholar, 39Kamimura Y. Masumoto H. Sugino A. Araki H. Mol. Cell. Biol. 1998; 18: 6102-6109Crossref PubMed Scopus (137) Google Scholar). The goal of this study was to characterize the Cdc7p/Dbf4p protein kinase and its role in chromosomal DNA replication initiation. Initially, attempts were made to purify the Cdc7p/Dbf4p complex fromS. cerevisiae cell extracts. However, because of its low abundance and the cell cycle-dependent expression of its activity, it was not possible to purify the complex to homogeneity. 2M. Kihara, W. Nakai, S. Asano, A. Suzuki, K. Kitada, Y. Kawasaki, L. H. Johnston, and A. Sugino, unpublished results. Thus, Cdc7p or Dbf4p were expressed in insect cells using the baculovirus system. Although insect cells expressed each protein, the majority of the protein was insoluble (data not shown). Attempts to reconstitute a soluble Cdc7/Dbf4 complex by mixing two cell extracts containing Cdc7p and Dbf4p did not produce any significant amount of soluble, active Cdc7p/Dbf4p complex. Therefore, Cdc7p and Dbf4p were co-expressed in insect cells by mixed infection using the baculovirus system. When soluble extracts were prepared from these cells and precipitated with rabbit antiserum against Cdc7p, both Cdc7p and Dbf4p were precipitated efficiently (see below). Furthermore, the immunoprecipitates incorporated a significant amount of 32P into a GST-Mcm2p substrate when co-incubated with the substrate and [γ-32P]ATP. The Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells expressing both Cdc7p and Dbf4p as described under “Materials and Methods.” Fig. 1 shows the elution profile of the partially purified protein complex when applied to a Hi Trap Heparin column, eluted from the column with a 0.2–1 m NaCl linear gradient, and assayed for protein kinase activity using GST-Mcm2p as a substrate. Two peaks of kinase activity, which were not detected in uninfected insect cells (data not shown), are observed; the first peak coincides with peaks of both Cdc7p and Dbf4p (as detected by immunoblotting), suggesting that this peak represent the Cdc7p/Dbf4p protein kinase complex. On the other hand, the second activity peak did not include a significant amount of Dbf4p polypeptide, although it did include some Cdc7p polypeptide (Fig. 1). One explanation for this result is that the second activity peak might coincide with the elution position of an insect cell Dbf4 homologue. Alternatively, Cdc7p might be activated by unknown factor(s). For the purpose of this study, the fractions in the first peak of kinase activity were pooled and further purified by chromatography using several columns (see “Materials and Methods”). The product of the purification is nearly homogeneous and includes two major polypeptides (85 and 58 kDa, respectively) in nearly equimolar quantities. These polypeptides match the expected sizes of Dbf4p and Cdc7p and react appropriately with Cdc7p and Dbf4p antibodies (Fig.1 D). Therefore, we concluded that these two polypeptides are Dbf4p and Cdc7p, respectively. We designate here the Cdc7p/Dbf4p complex purified from insect cells as Cdc7p/Dbf4p* until it will be found that the Cdc7p/Dbf4p protein kinase complex from insect cells is the same as the complex from yeast cells. The untagged and soluble form of the Cdc7p/Dbf4p* protein kinase (Fig. 1) was used for extensive biochemical characterization. Previous work (31Lei M. Kawasaki Y. Young M.R. Kihara M. Sugino A. Tye B.K. Genes Dev. 1997; 11: 3365-3374Crossref PubMed Scopus (244) Google Scholar) demonstrated that the best substrate for the Cdc7p/Dbf4p* protein kinase is GST-Mcm2p purified from yeast cells, so this protein was used as a substrate in many of the following experiments. As shown in Fig. 2, the Cdc7p/Dbf4p* protein kinase quickly phosphorylated GST-Mcm2p (Fig. 2,closed circles) and autophosphorylated both Dbf4p (Fig. 2,open squares) and Cdc7p (data not shown) in 10 min at 30 °C. These data were used to determine that theV max of the kinase activity is 14.5 mol of phosphate/mol of Cdc7/Dbf4p* complex/min at 30 °C. However, theK m for the substrate could not be determined because only 0.2 mol of phosphate was incorporated per mole of substrate, which is most likely because the substrate is not 100% active. As found for most other protein kinases, the activity requires 5–10 mm Mg2+; 1–5 mm Zn2+or Ca2+ partially substitutes for Mg2+ giving 40 and 55% maximal activity, respectively. As shown in Fig.3 A, an addition of 0.2m NaCl severely inhibited phosphorylation of the GST-Mc" @default.
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• W2061065780 date "2000-11-01" @default.
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• W2061065780 title "Characterization of the Yeast Cdc7p/Dbf4p Complex Purified from Insect Cells" @default.
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