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• W1987293272 abstract "The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle and cyclin-dependent kinase (CDK) plays an important role in this process. Adenine nucleotides that bind to the origin recognition complex (ORC) are also suggested to be involved in this process. Of the six subunits of the Saccharomyces cerevisiae ORC (Orc1–6p), both Orc1p and Orc5p have ATP binding activity, and both Orc2p and Orc6p are phosphorylated by CDK in cells. In this study we constructed a series of yeast strains expressing phospho-mimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-S transition and S phase progression and causes the accumulation of cells with 2C DNA content. Using antibody that specifically recognizes Ser-188-phosphorylated Orc2p, we showed that Ser-188 is phosphorylated by CDK in a cell cycle-regulated manner. Expression of Orc2-5Dp caused phosphorylation of Rad53p and inefficient loading of the six minichromosome maintenance proteins. These results suggest that the accumulation of cells with 2C DNA content is due to inefficient origin firing and induction of the cell cycle checkpoint response and that dephosphorylation of Ser-188 of Orc2p in late M or G1 phase may be involved in pre-RC formation. In vitro, a purified mutant ORC containing Orc2-5Dp lost Orc5p ATP binding activity. This is the first demonstration of a link between phosphorylation of the ORC and its ability to bind ATP, which may be important for the cell cycle-regulated initiation of DNA replication. The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle and cyclin-dependent kinase (CDK) plays an important role in this process. Adenine nucleotides that bind to the origin recognition complex (ORC) are also suggested to be involved in this process. Of the six subunits of the Saccharomyces cerevisiae ORC (Orc1–6p), both Orc1p and Orc5p have ATP binding activity, and both Orc2p and Orc6p are phosphorylated by CDK in cells. In this study we constructed a series of yeast strains expressing phospho-mimetic mutants of Orc2p or Orc6p and found that expression of a Ser-188 mutant of Orc2p (Orc2-5Dp) delays G1-S transition and S phase progression and causes the accumulation of cells with 2C DNA content. Using antibody that specifically recognizes Ser-188-phosphorylated Orc2p, we showed that Ser-188 is phosphorylated by CDK in a cell cycle-regulated manner. Expression of Orc2-5Dp caused phosphorylation of Rad53p and inefficient loading of the six minichromosome maintenance proteins. These results suggest that the accumulation of cells with 2C DNA content is due to inefficient origin firing and induction of the cell cycle checkpoint response and that dephosphorylation of Ser-188 of Orc2p in late M or G1 phase may be involved in pre-RC formation. In vitro, a purified mutant ORC containing Orc2-5Dp lost Orc5p ATP binding activity. This is the first demonstration of a link between phosphorylation of the ORC and its ability to bind ATP, which may be important for the cell cycle-regulated initiation of DNA replication. The initiation of chromosomal DNA replication is tightly regulated to replicate the genome just once per cell cycle. To achieve this, both induction of initiation at the G1-S boundary and inhibition of initiation in other phases of the cell cycle are required. The mechanisms governing this regulation in eukaryotes have been studied the most extensively in budding yeast (Saccharomyces cerevisiae), and we describe mostly events in budding yeast in this paper otherwise noticed. Cyclin-dependent protein kinases (CDKs) 2The abbreviations used are: CDK, cyclin-dependent protein kinase; ORC, origin recognition complex; pre-RC, pre-replicative complex; MCM, minichromosome maintenance proteins; 5-FOA, 5-fluoroorotic acid; HA, hemagglutinin; α-factor, α-mating factor; ChIP, chromatin immunoprecipitation; ARS1, autonomously replicating sequence 1; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter. 2The abbreviations used are: CDK, cyclin-dependent protein kinase; ORC, origin recognition complex; pre-RC, pre-replicative complex; MCM, minichromosome maintenance proteins; 5-FOA, 5-fluoroorotic acid; HA, hemagglutinin; α-factor, α-mating factor; ChIP, chromatin immunoprecipitation; ARS1, autonomously replicating sequence 1; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter. play essential roles in both the induction and inhibition of initiation; low CDK activity in late M and G1 phases is required to prepare for initiation of DNA replication, and high CDK activity in S, G2, and early M phases is required for suppression of re-initiation of DNA replication before cell division. This high CDK activity is also involved in initiation of DNA replication at the G1-S boundary (1Kelly T.J. Brown G.W. Annu. Rev. Biochem.. 2000; 69: 829-880Google Scholar, 2Bell S.P. Dutta A. Annu. Rev. Biochem.. 2002; 71: 333-374Google Scholar, 3Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol.. 1997; 13: 293-332Google Scholar, 4Diffley J.F. Curr. Biol.. 2004; 14: 778-786Google Scholar). Cell cycle-regulated formation of protein complexes on origins of chromosomal DNA replication is a key step in regulation of the initiation of DNA replication. In G1 phase (under low CDK activity), a protein complex called the “pre-replication complex (pre-RC)” is formed on each origin. The pre-RC contains several proteins including the origin recognition complex (ORC), Cdc6p, Cdt1p, and the six minichromosome maintenance proteins (MCM), Mcm2–7p. The ORC was originally identified as a six-protein complex that specifically bound to S. cerevisiae origins of DNA replication (5Bell S.P. Stillman B. Nature.. 1992; 357: 128-134Google Scholar), and its homologues have been found in various eukaryotic species, including human (3Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol.. 1997; 13: 293-332Google Scholar). In this manuscript, “ORC” refers to S. cerevisiae ORC. The ORC is bound to chromatin at the origins of chromosomal DNA replication throughout the cell cycle and is thought to function as a “landing pad” for the assembly of pre-RC. At the G1-S boundary, CDK and another kinase (Cdc7p-Dbf4p) activate the pre-RC to initiate chromosomal DNA replication. After initiation, re-formation of the pre-RC is strictly prohibited to suppress re-initiation of DNA replication, and high CDK activity is essential for this process; artificial inhibition of CDK activity in G2 phase resulted in re-formation of pre-RC and re-initiation of DNA replication (6Dahmann C. Diffley J.F. Nasmyth K.A. Curr. Biol.. 1995; 5: 1257-1269Google Scholar, 7Noton E. Diffley J.F. Mol. Cell.. 2000; 5: 85-95Google Scholar, 8Weinreich M. Liang C. Chen H.H. Stillman B. Proc. Natl. Acad. Sci. U. S. A.. 2001; 98: 11211-11217Google Scholar). The B type cyclin-CDK complex affects initiation of DNA replication through two distinct mechanisms, phosphorylation of, or direct binding to replication-related proteins (9Mimura S. Seki T. Tanaka S. Diffley J.F. Nature.. 2004; 431: 1118-1123Google Scholar, 10Wilmes G.M. Archambault V. Austin R.J. Jacobson M.D. Bell S.P. Cross F.R. Genes Dev.. 2004; 18: 981-991Google Scholar). Therefore, identification of the components of the protein complex present on origin DNA that are phosphorylated by CDK and an understanding of the role of this phosphorylation are important for understanding the mechanisms which ensure that replication occurs just once per cell cycle. It has been suggested that Orc2p, Orc6p, Cdc6p, and MCM are phosphorylated by CDK in a cell cycle-regulated manner. Phosphorylation of Cdc6p or MCM seems to cause its degradation or nuclear exclusion, respectively (11Elsasser S. Chi Y. Yang P. Campbell J.L. Mol. Biol. Cell.. 1999; 10: 3263-3277Google Scholar, 12Sanchez M. Calzada A. Bueno A. J. Biol. Chem.. 1999; 274: 9092-9097Google Scholar, 13Drury L.S. Perkins G. Diffley J.F. EMBO J.. 1997; 16: 5966-5976Google Scholar, 14Drury L.S. Perkins G. Diffley J.F. Curr. Biol.. 2000; 10: 231-240Google Scholar, 15Nguyen V.Q. Co C. Irie K. Li J.J. Curr. Biol.. 2000; 10: 195-205Google Scholar, 16Liku M.E. Nguyen V.Q. Rosales A.W. Irie K. Li J.J. Mol. Biol. Cell.. 2005; 16: 5026-5039Google Scholar, 17Labib K. Diffley J.F. Kearsey S.E. Nat. Cell Biol.. 1999; 1: 415-422Google Scholar). Furthermore, the expression of degradation-resistant Cdc6p and Mcm7p with an exogenous nuclear localization signal with the expression of mutant forms of Orc2p and Orc6p in which possible CDK-phosphorylated sites are mutated to be inert, induced reformation of the pre-RC and re-initiation of DNA replication without inhibition of CDK (18Nguyen V.Q. Co C. Li J.J. Nature.. 2001; 411: 1068-1073Google Scholar, 19Archambault V. Ikui A.E. Drapkin B.J. Cross F.R. Mol. Cell. Biol.. 2005; 25: 6707-6721Google Scholar). These results suggest that CDK-dependent phosphorylation of ORC, Cdc6p, and MCM play an important role in suppression of re-formation of pre-RC and re-initiation of DNA replication. However, at present it is unclear which subunit (Orc2p or Orc6p) or which possible CDK-phosphorylated site is responsible for this regulation and how the phosphorylation of ORC suppresses the re-initiation of DNA replication. Furthermore, the role of dephosphorylation of ORC in late M or G1 phase cannot be ruled out by use of mutants that are inert for phosphorylation. To address these issues, characterization of phospho-mimetic mutants (in which particular CDK target amino acid residues are substituted with Asp or Glu) is useful. For example, analysis of a phospho-mimetic mutant of Sld2p suggested that phosphorylation of Sld2p is responsible for CDK-dependent initiation of DNA replication (20Tanaka S. Umemori T. Hirai K. Muramatsu S. Kamimura Y. Araki H. Nature.. 2007; 445: 328-332Google Scholar, 21Tak Y.S. Tanaka Y. Endo S. Kamimura Y. Araki H. EMBO J.. 2006; 25: 1987-1996Google Scholar, 22Masumoto H. Muramatsu S. Kamimura Y. Araki H. Nature.. 2002; 415: 651-655Google Scholar). As is the case for the bacterial initiator of chromosomal DNA replication, DnaA (23Sekimizu K. Bramhill D. Kornberg A. Cell.. 1987; 50: 259-265Google Scholar, 24Mizushima T. Sasaki S. Ohishi H. Kobayashi M. Katayama T. Miki T. Maeda M. Sekimizu K. J. Biol. Chem.. 1996; 271: 25178-25183Google Scholar, 25Mizushima T. Takaki T. Kubota T. Tsuchiya T. Miki T. Katayama T. Sekimizu K. J. Biol. Chem.. 1998; 273: 20847-20851Google Scholar, 26Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell.. 1998; 94: 61-71Google Scholar, 27Mizushima T. Nishida S. Kurokawa K. Katayama T. Miki T. Sekimizu K. EMBO J.. 1997; 16: 3724-3730Google Scholar), adenine nucleotides bound to the ORC seem to regulate the initiation of DNA replication. The ORC has two subunits (Orc1p and Orc5p), which bind ATP (28Klemm R.D. Austin R.J. Bell S.P. Cell.. 1997; 88: 493-502Google Scholar, 29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar). Orc1p, but not Orc5p, has ATPase activity, which stimulates the loading of MCM onto origins (30Bowers J.L. Randell J.C. Chen S. Bell S.P. Mol. Cell.. 2004; 16: 967-978Google Scholar, 31Randell J.C. Bowers J.L. Rodriguez H.K. Bell S.P. Mol. Cell.. 2006; 21: 29-39Google Scholar). Orc5p, but not Orc1p, can bind ADP (32Takenaka H. Makise M. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Mol. Biol.. 2004; 340: 29-37Google Scholar). The binding of ATP to Orc1p, but not to Orc5p, is essential for the specific binding of ORC to origin DNA (28Klemm R.D. Austin R.J. Bell S.P. Cell.. 1997; 88: 493-502Google Scholar, 29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar). ATP binding to Orc5p increases the affinity of Orc1p for ATP in vitro (29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar) and is important for maintaining the stability of ORC in vivo (33Takahashi N. Yamaguchi Y. Yamairi F. Makise M. Takenaka H. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2004; 279: 8469-8477Google Scholar, 34Makise M. Takahashi N. Matsuda K. Yamairi F. Suzuki K. Tsuchiya T. Mizushima T. Biochem. J.. 2007; 402: 397-403Google Scholar). However, a link between phosphorylation of ORC and its ATP binding activity has not been previously established. In this study we constructed a series of yeast strains expressing phospho-mimetic mutant Orc2p or Orc6p for each possible CDK-phosphorylated site. We found that expression of a Ser-188 mutant of Orc2p, but not of other CDK-phosphorylated site mutants, delays cell growth, G1-S transition, S phase progression, and pre-RC formation. This suggests that Ser-188 is the key amino acid residue in CDK-dependent regulation of ORC. Thus, dephosphorylation of Ser-188 of Orc2p in late M or G1 phase may be involved in the formation of pre-RC. In vitro, the purified phospho-mimetic mutant ORC containing the Ser-188 Orc2p mutant retained the ATP binding activity of Orc1p and origin DNA binding activity but lost the ATP binding activity of Orc5p. We consider that phosphorylation of Ser-188 of Orc2p affects pre-RC formation by inhibiting the binding of ATP to Orc5p. Chemicals, Plasmids, and Strains—[α-32P]ATP (3000 Ci/mmol), [γ-32P]ATP (6000 Ci/mmol), and poly(dI/dC) were purchased from GE Healthcare, and 8-N3-[γ-32P]ATP was from ALT Bioscience. DNA fragments (290 bp) containing wild-type ARS1 and mutant ars1/A–B1– were prepared by PCR as described previously (35Mizushima T. Takahashi N. Stillman B. Genes Dev.. 2000; 14: 1631-1641Google Scholar) and purified by polyacrylamide gel electrophoresis. DNA fragments were radiolabeled by T4 polynucleotide kinase and [γ-32P]ATP as described previously (29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar). The specific activity of each probe was ∼4000 cpm/fmol DNA. Mouse monoclonal antibodies against Orc3p, Mcm2p, and hemagglutinin (HA) were gifts from Dr. Stillman (Cold Spring Harbor Laboratory). Mouse monoclonal antibody against FLAG was purchased from Sigma. Plasmids, pRS403, 405, 413, 416, and 425 were purchased from Invitrogen. Plasmids pRS413-ORC2 and pRS416-ORC2 contain ORC2 (from –805 to +1909) ligated into the SacI-SalI site. To express HA-tagged Orc2p in cells, pRS413–3HA and pRS413-GAL1-3HA were first constructed. Plasmid pRS413–3HA has a triple HA epitope gene (3HA) in the XhoI-ApaI site, and pRS413-GAL1-3HA has the GAL1 promoter (from –457 to –1) in the BamHI-EcoRV site. An expression plasmid, pRS413-ORC2-3HA (or pRS413-GAL1-ORC2-3HA), contains ORC2 ligated in-frame with 3HA. A plasmid pRS425-GAL1-ORC2-3HA was constructed by insertion of the BssHII-BssHII DNA fragment of pRS413-GAL1-ORC2-3HA into the same restriction enzyme site on pRS425. Plasmids pRS413-ORC6 and pRS416-ORC6 contain ORC6 (from –600 to +1808) cloned into the XbaI-XhoI site. Site-directed mutagenesis of the CDK consensus sites was done as described previously (36Matsuda K. Makise M. Sueyasu Y. Takehara M. Asano T. Mizushima T. FEMS Yeast Res.. 2007; 7: 1263-1269Google Scholar). DNA fragments containing orc2- (or orc6)-All-A were PCR-amplified using chromosomal DNA prepared from pJL1095 and pJL1096 cells (gifts from Dr. Li (University of California, San Francisco)) (18Nguyen V.Q. Co C. Li J.J. Nature.. 2001; 411: 1068-1073Google Scholar) as templates. A series of expression plasmids for mutant forms of orc were constructed by replacement of the wild-type ORC gene with each mutant orc gene. S. cerevisiae strains are listed in Table 1 (37Thomas B.J. Rothstein R. Cell.. 1989; 56: 619-630Google Scholar, 38Liang C. Weinreich M. Stillman B. Cell.. 1995; 81: 667-676Google Scholar). These strains were cultured in YP medium (2% bactopeptone, 1% yeast extract, 2 mg/ml adenine hemisulfate) or synthetic complete medium containing glucose or galactose.TABLE 1Yeast strains used in this studyStrainsGenotypeReferencesW303-1AMATa leu2-3,112 ura3-52 can1-100 ade2-1 his3-11 trp1-1Ref. 37Thomas B.J. Rothstein R. Cell.. 1989; 56: 619-630Google ScholarDK186W303-1A bar1ΔRef. 57Harvey S.L. Kellogg D.R. Curr. Biol.. 2003; 13: 264-275Google ScholarYMM10DK186 orc2D::TRP1[pRS416-ORC2]This studyYMM10-2YMM10[pRS413]This studyYMM10-3YMM10[pRS413-ORC2]This studyYMM10-4YMM10[pRS413-orc2-All-D]This studyYMM10-5YMM10[pRS413-orc2-All-A]This studyYMM10-6YMM10[pRS413-orc2—2d]This studyYMM10-7YMM10[pRS413-orc2-12d]This studyYMM10-8YMM10[pRS413-orc2-123d]This studyYMM10-9YMM10[pRS413-orc2-4d]This studyYMM10-10YMM10[pRS413-orc2-5d]This studyYMM10-11YMM10[pRS413-orc2-6d]This studyYMM10-12YMM10[pRS413-orc2-56d]This studyYMM10-13YMM10[pRS413-orc2-456d]This studyYMM10-14YMM10[pRS413-orc2-12346d]This studyYMM10-15YMM10[pRS413-orc2-5a]This studyYMM18DK186 orc6Δ::TRP1[pRS416-ORC6]This studyYMM18-2YMM18[pRS413]This studyYMM18-3YMM18[pRS413-ORC6]This studyYMM18-4YMM18[pRS413-orc6-All-D]This studyYMM18-5YMM18[pRS413-orc6-All-A]This studyYMM69YMM10 [pRS425-GAL1-ORC2-3HA]This studyYMM71-1YMM10 [pRS425-GAL1-orc2-5d-3HA]This studyYMM71-2YMM10 [pRS425-GAL1-orc2-5a-3HA]This studyYMM76YMM77 his3::pRS403-orc2-5d-3HAThis studyYMM77DK186 orc2Δ::TRP1 leu2::pRS405-GAL1-orc2-1-3FLAGThis studyYMM84YMM77 his3::pRS403-ORC2-3HAThis studyYMM87W303-1A [pRS425-GAL1-ORC2-3HA]This studyYMM88W303-1A cdc28-4 [pRS425-GAL1-ORC2-3HA]This study Open table in a new tab To disrupt the chromosomal ORC2 or ORC6, TRP1 was inserted between flanking sequences (40 bp) of the ORC2 or ORC6, and this DNA fragment was introduced into the DK186 diploid (resultant strains were YMM10 or YMM18, respectively). We confirmed that all tetrads showed only two viable spores. For plasmid shuffling, plasmid pRS413 (a low copy number plasmid containing HIS) (39Sikorski R.S. Hieter P. Genetics.. 1989; 122: 19-27Google Scholar) with each mutant orc2 or orc6 gene, was transformed into YMM10 or YMM18 cells by the lithium acetate method. The transformants were cultured on synthetic complete agar plates containing 2% glucose and 0.1% 5-fluoroorotic acid (5-FOA) at 30 °C for 3 days. YMM77 is a derivative of YMM10 in which pRS405-GAL1-orc2-1–3FLAG was integrated into the leu2 locus. YMM84 and YMM76 are derivatives of YMM77 in which pRS403-ORC2-3HA and pRS403-orc2-5d-3HA, respectively, were integrated into the his3 locus. We confirmed these integrations by PCR and Southern blot analysis. Preparation of Antibodies and ORC—Rabbit polyclonal antibody against Ser-188-phosphorylated Orc2p (α-Ser(P)-188) was generated against synthetic peptide with the following sequence: NHDFTS(PO4)PLKQIIC (40Kuniyasu A. Kaneko K. Kawahara K. Nakayama H. FEBS Lett.. 2003; 552: 259-263Google Scholar). The antibody was purified on a protein A-Sepharose column followed by Sulfolink Coupling Gel (Pierce) coupled with the synthetic peptide. The specificity of the antibody was tested by enzyme-linked immunosorbent assay using the peptide and the peptide lacking the phosphorus at the serine residue (data not shown). ORCs were co-expressed in Sf9 cells infected with recombinant baculoviruses and purified as described (29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar). Baculovirus containing the phospho-mimetic orc2 gene was constructed using a BD BaculoGold™ transfection kit (BD Biosciences) according to the manufacturer's instructions. Phosphorylation of ORC—Purification of yeast recombinant CDK (rGST-Cdc28-Clb5) was performed as described (21Tak Y.S. Tanaka Y. Endo S. Kamimura Y. Araki H. EMBO J.. 2006; 25: 1987-1996Google Scholar). The plasmid, pGEX6P-1/CDC28-CAK1-CKS1-CLB5 was gift from Dr. Araki (National Institute of Genetics). To obtain an active recombinant GST-Cdc28/Clb5 complex (rGST-Cdc28-Clb5), the cell extracts prepared from Escherichia coli Rosseta 2(DE)pLysS cells (Novagen) harboring the plasmid was applied to the glutathione-Sepharose column, and rGST-Cdc28-Clb5 was eluted with the buffer containing reduced glutathione. For phosphorylation of ORC, ORC (30 pmol) and recombinant CDK (13.5 pmol) were incubated at 25 °C for 24 h in the buffer (600 μl) containing 50 mm Hepes-KOH, pH 7.5, 10 mm MgCl2, 1 mm ATP. To remove ATP from ORC samples, ORC was precipitated with SP-Sepharose (GE Healthcare) and eluted with the buffer containing 50 mm Hepes-KOH, pH 7.5, 500 mm KCl, 5 mm Mg(OAc)2, 1 mm EDTA, 1 mm EGTA, 0.02% v/v Nonidet P-40, and 10% v/v glycerol. UV-cross-linking Assay—UV-cross-linking experiments were done as described previously (28Klemm R.D. Austin R.J. Bell S.P. Cell.. 1997; 88: 493-502Google Scholar, 29Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2003; 278: 46440-46445Google Scholar). ORC (3 pmol) was incubated with 4 μm 8-N3-[γ-32P]ATP in the presence or absence of ARS1 DNA fragments at 4 °C for 5 min. Samples were placed on Parafilm and subjected to UV irradiation at 4 °C for 2 min. The photolabeling reaction was terminated by the addition of stop solution (70 μl) containing 0.1 m dithiothreitol and 20 mm EDTA. Samples were precipitated by 20% trichloroacetic acid, washed with acetone, and separated by electrophoresis on a polyacrylamide gel (10%) containing SDS. Gels were stained with silver to identify each ORC subunit, and radiolabeled subunits were detected by autoradiography. Filter Binding Assay—ORC was incubated with [α-32P]ATP or radiolabeled DNA fragments (200 fmol) at 30 °C for 5 min in 40 μl of buffer T (25 mm Tris-HCl, pH 7.6, 5 mm MgCl2, 70 mm KCl, 5 mm dithiothreitol, and 5% (v/v) glycerol). In some of the ATP binding experiments, ORC was further incubated with DNA fragments at 30 °C for 5 min in the same buffer. Samples were passed through nitrocellulose membranes (Millipore HA, 0.45 μm) and washed with 5 ml of ice-cold buffer T twice. The radioactivity remaining on the filter was monitored with a liquid scintillation counter. ATPase Assay—The ATPase activity of ORC was measured as described previously (28Klemm R.D. Austin R.J. Bell S.P. Cell.. 1997; 88: 493-502Google Scholar) with some modifications. ORC (0.3 pmol) was incubated with DNA fragments (6 pmol) in 10 μl of ATPase buffer (50 mm Hepes-KOH pH7.6, 150 mm KCl, 5 mm Mg(OAc)2, 1 mm EDTA, 1 mm EGTA, 0.02% v/v Nonidet P-40, and 10 μm radiolabeled ATP) for 60 min at room temperature. The reaction was stopped by the addition of 2% w/v SDS (5 μl), and adenine nucleotides were separated on polyethyleneimine cellulose F TLC plates (Merck). Chromatin Binding Assay—Yeast spheroplasts were lysed with Triton X-100, and the samples were processed into soluble (supernatant) and chromatin (insoluble precipitate) fractions by centrifugation as previously described (41Liang C. Stillman B. Genes Dev.. 1997; 11: 3375-3386Google Scholar, 42Takahashi N. Tsutsumi S. Tsuchiya T. Stillman B. Mizushima T. J. Biol. Chem.. 2002; 277: 16033-16040Google Scholar). Equivalent amounts (total protein) of chromatin fractions were subjected to electrophoresis on a polyacrylamide gel (10%) containing SDS, transferred to polyvinylidene difluoride membranes, and probed with antibodies. FACS Analysis—The samples were prepared as previously described (33Takahashi N. Yamaguchi Y. Yamairi F. Makise M. Takenaka H. Tsuchiya T. Mizushima T. J. Biol. Chem.. 2004; 279: 8469-8477Google Scholar) with some modifications. The cells were pelleted by centrifugation and fixed in 70% ethanol for 1 h. The cells were pelleted by centrifugation, washed with 50 mm sodium citrate once, pelleted again by centrifugation, incubated with 50 mm sodium citrate containing 0.25 mg/ml RNase A for 1 h at 50 °C, and then treated with 1 mg/ml proteinase K for 1 h at 50 °C. DNA was stained with 50 μg/ml propidium iodide at 4 °C for 1 h, and 20,000 cells from each sample were scanned with a FACSCalibur (BD Biosciences). Gel Electrophoretic Mobility Shift Assay—A gel electrophoretic mobility shift assay was performed as described (32Takenaka H. Makise M. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Mol. Biol.. 2004; 340: 29-37Google Scholar, 43Lee J.R. Makise M. Takenaka H. Takahashi N. Yamaguchi Y. Tsuchiya T. Mizushima T. Biochem. J.. 2002; 362: 395-399Google Scholar) with some modifications. ORCs were incubated with adenine nucleotides for 5 min at 30 °C and with radiolabeled wild-type ARS1 or mutant ars1/A–B1– DNA fragments (100 fmol) for 5 min at 30 °C in 10 μl of buffer T containing 2 mg/ml of bovine serum albumin and 10 μg/ml poly(dI/dC) (nonspecific competitors). The reaction sample was loaded onto a 3.5% polyacrylamide gel containing 0.5 × TBE (0.045 m Tris borate, pH 8.3, and 1 mm EDTA). The gel was electrophoresed for 1.5 h at a constant 200 V, dried, and autoradiographed. Chromatin immunoprecipitation (ChIP) Assay—A ChIP assay was done as described previously (44Aparicio O.M. Weinstein D.M. Bell S.P. Cell.. 1997; 91: 59-69Google Scholar) with some modifications. Cells were cross-linked with 1% formaldehyde for 15 min at 25 °C. After the addition of 125 mm (final concentration) glycine, cells were harvested and lysed with glass beads in the buffer (50 mm Hepes-KOH, pH 7.5, 140 mm NaCl, 1 mm EDTA, 1% Triton X-100 (v/v), 0.1% sodium deoxycholate (w/v), 1 mm phenylmethylsulfonyl fluoride, 2 mm benzamidine, 1 μg/ml leupeptin, and 2 μg/ml pepstatin A). Samples were sonicated 30 times for 10 s (to achieve an average fragment size of 0.5–1 kilobases). Immunoprecipitation was performed with magnetic beads which were coated with protein G (Dynal) and antibody against HA or Mcm2p. Precipitates were washed, processed for DNA purification, and subjected to PCR. The PCR cycles included an initial denaturation step of 0.5 min at 95 °C, which was followed by 35 cycles of a denaturation step for 0.5 min at 95 °C, an annealing step for 0.5 min at 50 °C, a polymerization step for 1 min at 72 °C, and a final extension for 4 min at 72 °C. The PCR products were separated on a 3% agarose gel and visualized under UV after ethidium bromide staining. Site-directed Mutational Analysis of Orc2p and Orc6p Phosphorylation—It was reported that Orc2p and Orc6p have six (Ser-16, Ser-24, Thr-70, Thr-174, Ser-188, and Ser-206) and four (Ser-106, Ser-116, Ser-123, and Thr-146) consensus CDK phosphorylation sites ((S/T)PX(K/R)), respectively (Fig. 1A). Expression of mutant forms of Orc2p and Orc6p in which these amino acid residues were substituted with Ala caused re-initiation of DNA replication in G2 phase when a degradation-resistant mutant of Cdc6p and a Mcm7p with an exogenous nuclear localization signal were expressed simultaneously (18Nguyen V.Q. Co C. Li J.J. Nature.. 2001; 411: 1068-1073Google Scholar, 19Archambault V. Ikui A.E. Drapkin B.J. Cross F.R. Mol. Cell. Biol.. 2005; 25: 6707-6721Google Scholar). We constructed a phospho-mimetic mutant of Orc2p, Orc2-All-Dp, in which all of the consensus CDK phosphorylation sites were mutated to Asp. Orc6-All-Dp was constructed in a similar way. We also constructed Orc2-All-Ap and Orc6-All-Ap in which the consensus CDK phosphorylation sites were mutated to Ala. Each mutant gene was inserted into a plasmid, and the plasmid was transformed into strain YMM10 (or YMM18) which contains a chromosomal ORC2 or ORC6 deletion and an alternative wild-type gene on a plasmid with the URA3 selectable maker. When the transformants were grown on agar plates containing 5-FOA, the URA3 plasmid was selected against and lost, causing cells to rely solely on the mutant orc2 or orc6 gene (plasmid-shuffling analysis). YMM10 (or YMM18) cells containing plasmid carrying the wild-type genes could grow, and those containing vector only could not grow on the 5-FOA-containing plates (Fig. 1B), showing that the plasmid-shuffling system works well. As shown in Fig. 1B, YMM10 cells expressing Orc2-All-Dp could grow on agar plates without 5-FOA but not on those with 5-FOA. On the other hand, YMM18 expressing Orc6-All-Dp could grow even on agar plates containing 5-FOA (Fig. 1B). Plasmid shuffling analysis also showed that both of the Ala substitution mutants can support cell growth (Fig. 1B), as described previously (18Nguyen V.Q. Co C. Li J.J. Nature.. 2001; 411: 1068-1073Google Scholar). Based on results in Fig. 1, we consider that dephosphorylation of Orc2p but not of Orc6p is required for cell growth and cell cycle progression. To identify the amino acid residue responsible for the phenotype exhibited by cells expressing Orc2-All-Dp, we constructed a series of mutant Orc2p, as shown in Fig. 1C, and analyzed the function of each mutant protein by plasmid shuffling analysis. Results show that the S188D Orc2p mutant (Orc2-5Dp) but not the variants of Orc2p with mutations in the other consensus CDK phosphorylation sites is unable to support cell growth (Fig. 1C). Furthermore, a mutant Orc2p in which all of the consensus CDK phosphorylation sites except the Ser-188 are replaced with Asp (Orc2-12346Dp) could support cell growth (Fig. 1C). Thus, we concluded that Ser-188 is an important target of CDK for controlling cell cycle progression, and phosphorylation of this amino acid residue may block cell cycle progression. To detect phosphorylation of Ser-188 of Orc2p in vivo, we prepared polyclonal antibody that recognizes Ser-188-phosphorylated Orc2p (α-Ser(P)-188). As shown in Fig. 2A, α-Ser(P)-188 detected a band with the same migration as that detected by antibody against HA in cells expressing wild-type HA-tagged Orc2p but not in cells expressing HA-tagged Ser-188 Orc2p mutant (Orc2-5Dp or Orc2-5Ap). The band was not detected in the wild-type Orc2p-containing sample that had been treated with λ-protein phosphatase (Fig. 2B). Based on these results, we conclude that α-Ser(P)-188 can specifically recognize Ser-188-phosphorylated Orc2p. As sho" @default.
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• W1987293272 title "Linkage between Phosphorylation of the Origin Recognition Complex and Its ATP Binding Activity in Saccharomyces cerevisiae" @default.
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