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• W2034156963 abstract "Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 °C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process. Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 °C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process. The initiation of chromosomal DNA replication is tightly regulated to achieve genome replication just once per cell cycle. In chromosomal DNA replication, adenine nucleotides bound to initiator proteins are involved in this regulation both in prokaryotes and eukaryotes. In Escherichia coli, the DnaA protein, the initiator of chromosomal DNA replication, has a high affinity for both ATP and ADP (1Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (345) Google Scholar). The ATP-DnaA complex is active for DNA replication, but the ADP-DnaA complex and nucleotide-free DnaA are inactive both in vivo and in vitro (1Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 2Mizushima T. Sasaki S. Ohishi H. Kobayashi M. Katayama T. Miki T. Maeda M. Sekimizu K. J. Biol. Chem. 1996; 271: 25178-25183Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 3Mizushima T. Takaki T. Kubota T. Tsuchiya T. Miki T. Katayama T. Sekimizu K. J. Biol. Chem. 1998; 273: 20847-20851Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). DnaA has intrinsic ATPase activity, and ATP bound to DnaA can be hydrolyzed to ADP. This hydrolysis inactivates DnaA, suppressing rereplication and thus overinitiation of DNA replication (4Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 5Mizushima T. Nishida S. Kurokawa K. Katayama T. Miki T. Sekimizu K. EMBO J. 1997; 16: 3724-3730Crossref PubMed Scopus (59) Google Scholar). Acidic phospholipids, such as cardiolipin, interact with conserved basic amino acid residues of DnaA and stimulate the release of ADP from the ADP-DnaA complex, resulting in reactivation of the complex (6Makise M. Mima S. Tsuchiya T. Mizushima T. J. Biol. Chem. 2001; 276: 7450-7456Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7Makise M. Mima S. Tsuchiya T. Mizushima T. J. Biol. Chem. 2000; 275: 4513-4518Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 8Hase M. Yoshimi T. Ishikawa Y. Ohba A. Guo L. Mima S. Makise M. Yamaguchi Y. Tsuchiya T. Mizushima T. J. Biol. Chem. 1998; 273: 28651-28656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar). In eukaryotes, the origin recognition complex (ORC) 1The abbreviations used are: ORC, origin recognition complex; SC, synthetic complete; FACS, fluorescence-activated cell sorter. 1The abbreviations used are: ORC, origin recognition complex; SC, synthetic complete; FACS, fluorescence-activated cell sorter. is a possible initiator of chromosomal DNA replication (10Stillman B. Science. 1996; 274: 1659-1664Crossref PubMed Scopus (428) Google Scholar, 11Diffley J.F. Genes Dev. 1996; 10: 2819-2830Crossref PubMed Scopus (211) Google Scholar, 12Bell S.P. Curr. Opin. Genet. Dev. 1995; 5: 162-167Crossref PubMed Scopus (38) Google Scholar, 13Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1383) Google Scholar). ORC was originally identified as a six-protein complex that specifically binds to the Saccharomyces cerevisiae origins of chromosomal DNA replication (14Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (981) Google Scholar). ORC homologues have been found in various eukaryotic species, including humans (15Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (339) Google Scholar). The following observations were reported for ORC in S. cerevisiae. ORC has at least two subunits (Orc1p and Orc5p) that bind to ATP. Orc1p, but not Orc5p have ATPase activity (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). ATP binding to Orc1p is essential for specific ORC binding to origin DNA (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The ATPase activity of Orc1p may suppress rereplication, as is the case with DnaA (17Lee D.G. Makhov A.M. Klemm R.D. Griffith J.D. Bell S.P. EMBO J. 2000; 19: 4774-4782Crossref PubMed Scopus (68) Google Scholar, 18Klemm R.D. Bell S.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8361-8367Crossref PubMed Scopus (67) Google Scholar). On the other hand, plasmid shuffling experiments for a mutant ORC, ORC5-A (containing Orc5p with a defective Walker A motif), revealed that cells expressing ORC5-A showed temperature-sensitive growth (19Loo S. Fox C.A. Rine J. Kobayashi R. Stillman B. Bell S.P. Mol. Biol. Cell. 1995; 6: 741-756Crossref PubMed Scopus (180) Google Scholar). This suggests that ATP binding to Orc5p is important for chromosomal DNA replication, but its precise role remains unknown. In this study, we examined the role of ATP binding to Orc5p in chromosomal DNA replication in cells by replacing the wild-type ORC5 gene on S. cerevisiae chromosome with the orc5-A gene to construct the orc5-A strain. We confirmed that this strain shows temperature-sensitive growth, which is suppressed by overproduction of the Orc4p subunit. Furthermore, we found that at high temperatures, the ORC5-A proteins become degraded. These results suggest that the binding of ATP to Orc5p is involved in the possibly stable association of Orc4p to the rest ORC subunits. Strains, Plasmids, and Medium—The S. cerevisiae strains are listed in Table I (20Thomas B.J. Rothstein R. Cell. 1989; 56: 619-630Abstract Full Text PDF PubMed Scopus (1327) Google Scholar, 21Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (308) Google Scholar). The cells were cultured in synthetic complete (SC) medium or YPD medium (1% yeast extract, 2% Bacto peptone, and 2% glucose).Table IYeast strainsStrainGenotypeReferenceW303-1AMataade2-1 can 1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1Ref. 20W303-1BMatα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1Ref. 20Y303DMata/α diploid, cross of W303-1A and W303-1BThis studyYY101Mata/αade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 ORC5/orc5Δ::LEU2This studyYY401Mataade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 orc5Δ::LEU2 [pRS416-ORC5]This studyYY411Mataade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 orc5-AThis studyJRY4292Mataade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 orc5-1Ref. 21 Open table in a new tab To disrupt the chromosomal ORC5 gene, the LEU2 gene was inserted between flanking sequences (70 bp) of the ORC5 gene. This DNA fragment was introduced into the W303 diploid (resultant strain, YY101). We confirmed that all tetrads showed only two viable spores. The ORC5 gene was amplified by PCR from chromosomal DNA of the W303-1A strain and inserted into pRS416 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), a low copy number plasmid containing the URA3 gene, to create pRS416-ORC5, which was used for the plasmid shuffling experiments. Site-specific mutation was performed using a PCR-mediated method (see Fig. 1). The orc5-A gene (orc5K43E) was introduced into pRS406 (another plasmid containing the URA3 gene) (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to create pRS406-orc5-A. This plasmid was transformed into W303-1A, and the transformed cells were selected on SC agar plates lacking uracil. The resultant strain was transferred to plates containing 5-fluoroorotic acid (the two-step gene replacement method (23Scherer S. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4951-4955Crossref PubMed Scopus (478) Google Scholar)). A colony whose growth was sensitive to high temperature was selected as the orc5-A strain (YY411). We confirmed that pRS416-ORC5 suppressed this temperature sensitivity. To overexpress each ORC subunit and Cdc6p in cells, the genes for these proteins were amplified by PCR from chromosomal DNA of the W303-1A strain and introduced to pRS426, a high copy number plasmid containing URA3 (24Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1416) Google Scholar); pRS416, a low copy number plasmid containing URA3 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar); YEplac181, a high copy number plasmid containing LEU2 (25Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2496) Google Scholar); or pRS415, a low copy number plasmid containing LEU2 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Plasmid Shuffling Analysis—The YY101 strain was transformed with pRS416-ORC5 and sporulated, and the spores were dissected. Ura+ Leu+ spores were selected (YY401) and used for plasmid shuffling experiments. Plasmid pRS414 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), a low copy number plasmid containing the TRP1 gene and containing each mutant orc5 gene, was transformed into YY401 by the lithium-acetate method. The transformant was selected on SC agar plates, containing 5-fluoroorotic acid, without tryptophan. Fluorescence-activated Cell Sorter (FACS) Analysis—The samples were prepared as previously described (26Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar) with the following modifications. The cells were pelleted by centrifugation, washed with sterilized water, and fixed in 70% ethanol for 12 h. The cells were again pelleted, resuspended in 50 mm sodium citrate, sonicated for 1 min, treated with 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 of propidium iodide, and 20,000 cells from each sample were scanned with a FACSCalibur (Becton Dickinson). Pulse Field Gel Electrophoresis—Pulse field gel electrophoresis experiments were as previously described (27Schwartz D.C. Cantor C.R. Cell. 1984; 37: 67-75Abstract Full Text PDF PubMed Scopus (2014) Google Scholar, 28Carle G.F. Olson M.V. Nucleic Acids Res. 1984; 12: 5647-5664Crossref PubMed Scopus (607) Google Scholar) with the following modifications. The cells were harvested by centrifugation and washed three times with solution I (50 mm Tris-HCl, 1.2 m sorbitol, 20 mm EDTA), resuspended in solution II (50 mm Tris-HCl, 1.2 m sorbitol, 20 mm EDTA, 5% β-mercaptoethanol), and incubated for 10 min at room temperature. The cells were then suspended in solution III (0.1 m sodium citrate/citric acid, 1.2 m sorbitol, 10 mm EDTA) and mixed with 2.4% agarose (low melt preparative grade; Bio-Rad). The resultant solidified blocks of agarose were transferred to solution IV (0.1 m sodium citrate/citric acid, 1.2 m sorbitol, 10 mm EDTA, 0.5% zymolyase) and incubated for 24 h at 37 °C. Blocks of agarose were washed with solution III and then with solution I once and incubated with solution V (0.1 m EDTA, 1% sodium lauroylsarcosine, 0.1% proteinase K) for 24 h at 50 °C. After washing with 0.2 m EDTA, the samples were applied to a 1.5% agarose slab and subjected to electrophoresis for 15.2 h at 300V, 10 °C with a 50-100-s switch interval. The gels were stained with ethidium bromide and observed under a UV illuminator. Construction of a Yeast Genomic Library and Screening for a Multi-copy Suppressor Gene for the Temperature Sensitivity of the orc5-A Strain—Total chromosomal DNA was extracted from W303-1A cells and partially digested by Sau3AI. DNA fragments of 4-10 kilobase pairs were purified by ultracentrifugation in the presence of CsCl and ligated into the BamHI site of YEplac181 (a high copy number plasmid containing the LEU2 gene). The resultant yeast genomic library was introduced into YY411 (orc5-A) cells by the lithium-acetate method, and temperature-resistant transformants were selected on SC agar plates at 37 °C. Chromatin Binding Analysis—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 (26Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar). Equivalent amounts (total protein) of chromatin fractions were subjected to electrophoresis on 7.5% or 10% polyacrylamide gels containing SDS, transferred to polyvinylidene difluoride membrane, and probed with monoclonal antibodies against Orc3p (SB3) and Orc5p (SB5) (26Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar, 29Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). ORC Purification—Wild-type ORC and ORC5-A were expressed in Sf9 cells infected with recombinant baculoviruses and purified as described (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). A recombinant baculovirus for ORC5-A was donated by Dr. Stephan P. Bell (MIT). Filter Binding Assay for DNA Binding to ORC—DNA fragments (290 bp) containing an origin of chromosome replication (ARS1) and its mutant (A-B1-) were prepared by PCR as described previously (29Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar) and purified by polyacrylamide gel electrophoresis. DNA fragments were radiolabeled by T4 polynucleotide kinase and [γ-32P]ATP. The specific activity of each probe was about 4000 cpm/fmol DNA. ORC (0.2 pmol) was incubated with ATP for 5 min at 37 °C in buffer T (25 mm Tris-HCl, pH 7.6, 5 mm MgCl2, 70 mm KCl, 5 mm dithiothreitol, and 5% glycerol) and further incubated with radiolabeled ARS1 DNA fragments (0.4 pmol) at 37 °C for 15 min in the same buffer. The samples were passed through nitrocellulose membranes (Millipore HA, 0.45 μm) and washed with ice-cold buffer T. The radioactivity remaining on the filter was monitored with a liquid scintillation counter. Site-directed Mutational Analysis for ATP Binding to Orc5p—S. cerevisiae Orc5p has a complete Walker A motif and an incomplete Walker B motif (Fig. 1). Based on sequence similarity to other ATP-binding proteins, in Orc5p, K43 in the Walker A motif seems to be important for ATP binding (30Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar), and this amino acid residue is conserved in Orc5p from various species (Fig. 1). To study ATP binding to Orc5p, we constructed a mutant at the Walker A motif, the orc5-A gene (orc5-K43E; Fig. 1). In vitro, ORC containing this mutation (ORC5-A) showed no ATP binding to Orc5p (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The Walker B motif consensus sequence is DEXY (where X is a hydrophobic amino acid residue and Y is an acidic amino acid residue) and the corresponding sequence of S. cerevisiae Orc5p is DGFD, which means that the motif is incomplete (Fig. 1). We also constructed two types of mutant orc5 gene at this site: orc5-B1 with AAFD and orc5-B2 with DEFD (Fig. 1). Each mutant orc5 gene was inserted into pRS414 (22Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), and the resultant plasmid was transformed into yeast strain YY401, whose chromosomal ORC5 gene had been deleted but which had a wild-type ORC5 gene on a plasmid with the URA3 selectable marker. When the transformant was grown on agar plates containing 5-fluoroorotic acid, the URA3 plasmid was selected against and lost, causing cells to rely solely on the mutant orc5 gene (plasmid shuffling analysis). The cells that expressed the Orc5-Ap did not grow on agar plates at 37 °C (Fig. 2A). At 24 °C, they grew slowly but could form colonies (Fig. 2A). Thus, the orc5-A mutation confers temperature-sensitive growth as reported previously (19Loo S. Fox C.A. Rine J. Kobayashi R. Stillman B. Bell S.P. Mol. Biol. Cell. 1995; 6: 741-756Crossref PubMed Scopus (180) Google Scholar). The sizes of colonies and doubling rates of cells that expressed Orc5-B1p or Orc5-B2p were indistinguishable from those of cells expressing wild-type Orc5p (Fig. 2A and data not shown). These results suggest that the incomplete Walker B motif of Orc5p is not important for the function of ORC in cells. To examine the effect of ORC5-A on chromosomal DNA replication, we replaced the chromosomal wild-type ORC5 gene with the orc5-A gene (YY411 strain). As shown in Fig. 2B, this strain also showed temperature-sensitive growth on agar plates. After 8 h of incubation at 37 °C, more than 80% of YY411 cells showed a large bud, suggesting that they were in late S phase or G2/M phase. We also determined the efficiency of colony formation in wild-type cells and in YY411 cells, at both 24 and 37 °C. As shown in Table II, in YY411 cells, the ratio of colony formation efficiency (37 °C/24 °C) was less than 1 × 10-5, confirming that this strain has temperature-sensitive growth.Table IIColony formation efficiency of YY411 strainStrain/plasmidRatio of colony formation efficiency (37 °C/24 °C)W303-1A0.76YY4110.32 × 10−5YY411/pNT2030.90YY411/pNT2130.44 × 10−1 Open table in a new tab Cell Cycle Progression in orc5-A Cells—To determine which phase of the cell cycle was blocked at high temperatures, YY411 cells were grown at 24 °C and then shifted to 37 °C, and their DNA content was determined by FACS analysis. Compared with wild type, the proportion of cells with nearly 2C DNA content increased over time (Fig. 3), suggesting that most of cells were blocked in late S phase or G2/M phase. To distinguish between these two possibilities, we performed pulse field gel electrophoresis. Chromosomal DNA from cells in S phase does not enter the gel because of a lack of condensation of chromatin and the presence of DNA replication intermediates, in contrast to DNA from cells in other phases of the cell cycle (31Dillin A. Rine J. Science. 1998; 279: 1733-1737Crossref PubMed Scopus (47) Google Scholar, 32Takahashi N. Tsutsumi S. Tsuchiya T. Stillman B. Mizushima T. J. Biol. Chem. 2002; 277: 16033-16040Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 33Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar). We confirmed that chromosomal DNA from cells in S phase (hydroxyurea-treated cells) but not that from cells in G2/M phases (nocodazole-treated cells) remained in the well (Fig. 4, compare lanes 2 and 3). In wild-type cells and YY411 cells cultured at 37 °C, some chromosomal DNA entered the gel (Fig. 4); therefore, it is not the case that almost all of the YY411 cells cultured at 37 °C are in the S phase. It is not clear that whether most of the cells are in the G2/M phase or whether they are mixture of cells in both the S and G2/M phases (see “Discussion”). Identification of a Multi-copy Suppressor Gene for orc5-A Temperature Sensitivity—To reveal the mechanism of temperature sensitivity (and hence the role of ATP binding to Orc5p in chromosomal DNA replication), we constructed a yeast genomic library with a high copy number plasmid (YEplac181) and searched for a gene that could suppress the temperature sensitivity of YY411. We obtained 20 independent temperature-resistant colonies. Restriction enzyme mapping and direct DNA sequencing revealed that most had a plasmid containing the wild-type ORC5 gene, and the rest had a plasmid containing full-length ORC4 and TIF3 genes and a part of the MMS1 and SGV1 genes (pNT201; Fig. 5A). Of these, it is the ORC4 gene that suppressed temperature sensitivity. A plasmid that contains only the ORC4 gene (pNT204) suppressed temperature sensitivity, and deletion of the ORC4 gene from pNT201 (pNT205) diminished the activity of this plasmid for suppression (Fig. 5). As shown in Table II, in YY411, introduction of plasmid expressing the ORC4 gene restored the ratio of colony formation efficiency (37 °C/24 °C) to that of the wild-type strain. The ORC4 gene suppressed temperature sensitivity when present in a high copy number plasmid (YEplac181) and also to a lesser degree when present in a low copy number plasmid (pRS415; Table II). Furthermore, introduction of ORC4 restored cell cycle progression; FACS analysis revealed no accumulation of cells with nearly 2C DNA content after 37 °C culture (Fig. 3). Although introduction of the vector (YEplac181) into YY411 caused a slight increase in cells with 1C DNA content after 10 h of incubation at 37 °C, there is a significant difference between YY411 with the vector and YY411 with pNT203, in relation to the extent of accumulation of cells with nearly 2C DNA content (Fig. 3). We confirmed by immunoblotting analysis that introduction of pNT203 caused overproduction of Orc4p (data not shown). Therefore, we concluded that overproduction of Orc4p suppresses the temperature sensitivity of YY411 strain. To test whether the suppression is specific for the ORC4 gene, we also examined the effect of overproduction of each other ORC subunit. As predicted, the introduction of a plasmid containing ORC5 also suppressed temperature sensitivity, but introduction of plasmids expressing ORC1, ORC2, ORC3, or ORC6 did not (Fig. 6). We confirmed by immunoblotting analysis that each subunit was overproduced (data not shown). Therefore, suppression of temperature sensitivity is specific for the ORC4 gene, suggesting that Orc4p specifically affects the function of Orc5p. Comparison of the orc5-A Strain with Another Temperature-sensitive orc5 Mutant, the orc5-1 Strain—The orc5-1 strain is another well known temperature-sensitive orc5 mutant. As shown in Fig. 7A, introduction of a plasmid expressing the ORC4 gene (pNT203) did not suppress the temperature sensitivity of the orc5-1 strain (JRY4249). We confirmed by immunoblotting analysis that Orc4p was overproduced similarly both in YY411 and JRY4249 cells (data not shown). Overproduction of Cdc6p was reported to suppress the temperature sensitivity of the orc5-1 strain (21Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (308) Google Scholar). Cdc6p directly binds to ORC in vitro (21Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 29Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). We examined the effect of overproduction of Cdc6p on the temperature sensitivity of the orc5-A strain, using a high copy number plasmid with CDC6 gene (pRS426-CDC6). As shown in Fig. 7B, the introduction of pRS426-CDC6 suppressed the temperature sensitivity of the orc5-1 strain (JRY4249) but not that of the YY411 strain. We confirmed by immunoblotting analysis that Cdc6p was overproduced similarly in both strains (data not shown). In JRY4249, the extent of the suppression was much the same as reported previously (21Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (308) Google Scholar). Thus, the orc5-1 mutation affects the function of Orc5p differently from the orc5-A mutation. Although the orc5-1 strain has been used in many previous studies, the position of its mutation was not known. We sequenced the orc5-1gene and found only one amino acid substitution, L331P, which is located outside the ATP-binding region (Fig. 1). Leu331 is conserved among various species (34Quintana D.G. Thome K.C. Hou Z.H. Ligon A.H. Morton C.C. Dutta A. J. Biol. Chem. 1998; 273: 27137-27145Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Ishiai M. Dean F.B. Okumura K. Abe M. Moon K.Y. Amin A.A. Kagotani K. Taguchi H. Murakami Y. Hanaoka F. O'Donnell M. Hurwitz J. Eki T. Genomics. 1997; 46: 294-298Crossref PubMed Scopus (38) Google Scholar, 36Springer J. Kneissl M. Putter V. Grummt F. Chromosoma. 1999; 108: 243-249Crossref PubMed Scopus (10) Google Scholar), suggesting that it is important for the function of ORC. Because the temperature sensitivity of the orc5-1 strain was suppressed by overproduction of Cdc6p, it seems that Leu331 is involved in ORC binding to Cdc6p. Immunoblotting Analysis of the Levels and Location of ORC in Cells—The amount of chromatin-bound ORC5-A in YY411 cells at 37 °C was examined by use of a chromatin binding assay (26Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar). As shown in Fig. 8A, the amount of Orc5p in chromatin decreased after the incubation temperature was shifted from 24 to 37 °C. The amount of another subunit of ORC, Orc3p, in chromatin also decreased after the temperature shift (Fig. 8A), suggesting that the entire ORC5-A was decreased. Introduction of the plasmid overexpressing ORC4 partially restored the levels of Orc5p and Orc3p in chromatin at 37 °C (Fig. 8A). To test whether the temperature shift translocates ORC5-A from chromatin to other locations, we measured the amounts of Orc5p and Orc3p in non-chromatin-soluble fractions (the supernatants of centrifugation from chromatin precipitation). As shown in Fig. 8A, in YY411, these amounts also decreased after the temperature shift, suggesting that the total amount of ORC protein decreased, possibly as it became sensitive to protein degradation. To test this possibility, protein synthesis in YY411 and the wild-type cells was blocked by cycloheximide before temperature shift, and then the levels of Orc5p and Orc3p in chromatin were monitored. As shown in Fig. 8B, the amounts of Orc3p and Orc5p in chromatin again decreased, suggesting that ORC5-A becomes degraded at nonpermissive temperatures in cells. It was possible that instability of ORC-5A in YY411 at 37 °C is due to the cell cycle being arrested in the late S or G2/M phase under the conditions (Fig. 3). For example, if ORC (not only ORC5-A but also wild-type ORC) becomes unstable at S or G2/M phase, the results in Fig. 8 (A and B) can be explained. To test this possibility, we examined the stability of wild-type ORC in W303-1A and ORC5-A in YY411 with pNT203 after blocking cell cycle by nocodazole (G2/M phase) or hydroxyurea (S phase). As shown in Fig. 8C, blocking of cell cycle by these chemicals did not affect the stability of wild-type ORC in W303-1A and ORC5-A in YY411 with pNT203 at 37 °C. This result suggests that the instability of ORC5-A in YY411 at 37 °C is not due to the cell cycle arrest under these conditions. Biochemical Analysis on Stability of ORC5-A in Vitro—Previous biochemical studies revealed that ORC5-A maintains its DNA binding activity and ATP binding to Orc1p but not ATP binding to Orc5p (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), and we confirmed those findings here (data not shown). Here, we compare the stability (susceptibility to denaturation) in vitro of purified ORC5-A to that of purified wild-type ORC by a filter binding assay. Based on results in Fig. 9A, we concluded that wild-type ORC and ORC5-A binds to wild-type ARS1 DNA fragments in a sequence-specific manner in the presence of 0.32 μg of poly(dI/dC) (nonspecific competitor DNA). We also confirmed that both wild-type ORC and ORC5-A did not bind to mutant ARS1 DNA fragments (A-B1-) even after 37 °C incubation in the presence of 0.32 μg of poly(dI/dC) (Fig. 9A). Both wild-type ORC and ORC5-A were incubated at 37 °C, and then the remaining origin DNA binding activity was measured by a filter binding assay. As shown in Fig. 9B, both ORC and ORC-5A lost their DNA binding activity at approximately same rate. We also compared the stability at 37 °C of the complex of ORC5-A with DNA and the complex of wild-type ORC with DNA. As shown in Fig. 9C, both of the complexes were very stable at 37 °C. In this study, we showed that the YY411 strain expressing ORC5-A (ORC with Orc5pK43E, a mutation in the ATP-binding domain) showed temperature-sensitive growth, and we found that the amount of ORC is decreased at a nonpermissive temperature (37 °C). Experiments with cycloheximide suggested that at 37 °C in cells ORC5-A is more sensitive to degradation than wild-type ORC. Based on these results we consider that ATP binding to Orc5p is important to form the correct higher ordered structure of ORC. Suppression of the temperature sensitivity by overproduction of Orc4p was specific for the orc5-A strain. Orc4p did not suppress the temperature sensitivity of the orc5-1 strain, which has a mutation in Leu331, outside of the ATP-binding domain. Furthermore, overproduction of other subunit of ORC (Orc1p, Orc2p, Orc3p, or Orc6p) did not suppress the temperature sensitivity of the orc5-A strain. Therefore, Orc4p seems to be specifically involved in ATP binding to Orc5p itself or its function in DNA replication. There are two possible mechanisms. In ORC bound onto origin DNA, Orc4p and Orc5p either interact directly or are closely located on origin DNA (37Lee D.G. Bell S.P. Mol. Cell. Biol. 1997; 17: 7159-7168Crossref PubMed Scopus (166) Google Scholar). One possibility is that Orc5p interacts with Orc4p depending on ATP binding to Orc5p. Higher amounts of Orc4p (from a high copy number plasmid) may enable Orc4p to interact with Orc5p even in the absence of ATP binding to Orc5p. However, in glutathione S-transferase pull-down experiments for measuring the interaction of Orc5p with Orc4p, there was no difference between wild-type Orc5p and Orc5pK43E. 2N. Takahashi and T. Mizushima, unpublished results. Therefore, some other factors seem to be involved in the ATP binding-dependent Orc5p-Orc4p interaction of Orc5p. In previous UV cross-linking experiments, Orc4p was cross-linked with radiolabeled ATP analogue depending on ATP binding to Orc1p, suggesting that Orc4p is located close to the ATP-binding site of Orc1p and therefore that Orc4p interacts with Orc1p depending on ATP binding to Orc1p (16Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Furthermore, we recently found that ATP binding to Orc5p increases the affinity of ATP binding to Orc1p; the Kd value of ORC5-A for ATP is much higher than that for wild-type ORC (38Makise M. Takenaka H. Kuwae W. Takahashi N. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 46440-46445Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Therefore, it is also possible that, in ORC5-A, the absence of ATP binding to Orc5p may prevent ATP binding to Orc1p, which in turn decreases the interaction of Orc1p with Orc4p, resulting in ORC degradation. Higher amounts of Orc4p (from a high copy number plasmid) may enable Orc1p to interact with Orc4p even in the absence of ATP binding to Orc5p and Orc1p. FACS analysis showed that incubation of the orc5-A strain at nonpermissive temperatures caused accumulation of cells with nearly 2C DNA content. Pulse field gel electrophoresis suggested either that most cells are in G2/M phase or that there is a mixture of cells in S or G2/M. We think the latter more likely, for the following reasons. When YY411 cells were blocked at G2/M phase by nocodazole, incubated at 37 °C for several hours, and then released to medium containing α-factor (G1 arrest), the loading of the six minichromosome maintenance proteins onto chromatin was inhibited compared with the wild-type cells (data not shown). Mutants cells expressing Cdc6p with defective ATPase activity showed a defect in S phase progression, which was due to inefficient loading of minichromosome maintenance proteins onto chromatin (32Takahashi N. Tsutsumi S. Tsuchiya T. Stillman B. Mizushima T. J. Biol. Chem. 2002; 277: 16033-16040Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 39Schepers A. Diffley J.F. J. Mol. Biol. 2001; 308: 597-608Crossref PubMed Scopus (41) Google Scholar). When minichromosome maintenance protein was loaded onto a smaller fraction of origins of DNA replication than normal, DNA duplication should be prolonged, resulting in the accumulation of cells in the S phase. A similar mechanism may happen in YY411 cells at 37 °C. It is also possible that the S phase check point system is induced in YY411 cells at 37 °C, resulting in cell cycle arrest at S phase. Therefore, we assume that some cells are in the S phase under those conditions (YY411 cells at 37 °C). On the other hand, most orc mutants show growth arrest at the G2/M phase (31Dillin A. Rine J. Science. 1998; 279: 1733-1737Crossref PubMed Scopus (47) Google Scholar, 40Bell S.P. Kobayashi R. Stillman B. Science. 1993; 262: 1844-1849Crossref PubMed Scopus (366) Google Scholar). It has been suggested that in the orc2-1 mutant, growth arrest at the G2/M phase is due to the DNA damage and spindle assembly checkpoint (41Garber P.M. Rine J. Genetics. 2002; 161: 521-534PubMed Google Scholar). Therefore, by analogy, it seems that the orc5-A strain may also have a defect in G2-M progression by a similar mechanism, and therefore, we assume that some cells are arrested in the G2/M phase. We thank Dr. Bruce Stillman (Cold Spring Harbor Laboratory) for providing antibodies against ORC and Dr. Stephan P. Bell (MIT) for providing a recombinant baculovirus for ORC5-A." @default.
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