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• W2017508741 abstract "Saccharomyces cerevisiaeCdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. Saccharomyces cerevisiaeCdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. cyclin-dependent kinase autonomously replicating sequence cell division cycle glutathioneS-transferase pulsed field gel electrophoresis phenylmethylsulfonyl fluoride replication complex kilobase pair(s) hemagglutinin low phosphate medium fluorescence-activated cell sorting Cell cycle regulation is a complicated but highly coordinated process. It has a conserved mechanism among eukaryotes from yeast to human. The primary control of the eukaryotic cell cycle is provided by a family of cyclin-dependent kinases (CDKs)1 and their associated cyclins, which regulate kinase activity. In unicellular yeast cells, a single prototype CDK gene, CDC28 in the budding yeastSaccharomyces cerevisiae or cdc2 + in the fission yeast Schizosaccharomyces pombe functions at different cell cycle stages. Different cyclins activate the same kinase as different points in the cycle. It is now known that the central control of cell cycle progression by CDK complexes is regulated positively and negatively to monitor each step of the progression (1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1769) Google Scholar,2Arellano M. Moreno S. Int. J. Biochem. Cell Biol. 1997; 29: 559-573Crossref PubMed Scopus (166) Google Scholar). This regulatory control, associated with checkpoints, orchestrates various types of cell cycle genes throughout the cell cycle. In S. cerevisiae, over 70 temperature-sensitive cell division cycle (cdc) mutations have been isolated that control events throughout the cell cycle (3Pringle J.R. Hartwell L.H. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1981: 97-142Google Scholar, 4Wheals A.E. The Yeasts. Academic Press, London1987: 284-390Google Scholar). One of them,CDC6, is required in the late G1 and S phases of the cell cycle. At the non-permissive temperature, the cdc6mutant cells show a DNA synthesis defect (5Culotti J. Hartwell L.H. Exp. Cell Res. 1971; 67: 389-401Crossref PubMed Scopus (130) Google Scholar). The cdc6mutants undergo increased chromosomal loss and hyper-recombination, suggesting a fairly direct role in DNA replication (6Hartwell L.H. Smith D. Genetics. 1985; 110: 381-395Crossref PubMed Google Scholar). Tandem copies of different ARSs added to the mini-chromosome can suppress mitotic loss in cdc6, but not in cdc7, cdc9,cdc16, or cdc17 mutants (7Hogan E. Koshland D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3098-3102Crossref PubMed Scopus (132) Google Scholar), suggesting a role at replication origins. The CDC6 mRNA fluctuates periodically throughout the cell cycle (8Zhou C. Jong A. J. Biol. Chem. 1990; 265: 19904-19909Abstract Full Text PDF PubMed Google Scholar, 9Piatti S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (333) Google Scholar, 10Zwerschke W. Rottjakob H.-W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar), and its nuclear entry is cell cycle-dependent (11Jong A.Y. Young M. Chen G.C. Zhang S.Q Chen C. DNA Cell Biol. 1996; 15: 883-895Crossref PubMed Scopus (19) Google Scholar). The 5′-untranslated region of the CDC6 gene is very similar to a group of cell cycle genes that are either precursor enzymes for DNA synthesis or are directly involved in DNA replication (12Zhou C. Jong A.Y. DNA Cell Biol. 1993; 12: 363-370Crossref PubMed Scopus (7) Google Scholar). The involvement of Cdc6 in DNA replication has also been supported by direct analysis of origin function in cdc6–1 mutants using two-dimensional gel method (13Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (308) Google Scholar). The studies suggest that the Cdc6 and Orc5 protein interact and determine the frequency of initiation of DNA replication in yeast. Genetic interaction between CDC6 and ORC6 has also been reported (14Li J.J. Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (362) Google Scholar). The S. pombe cdc18+ is homologous to S. cerevisiae CDC6 (15Kelly T.J. Martin G.S. Forsburg S.L. Stephen R.J. Russo A. Nurse P. Cell. 1993; 74: 371-382Abstract Full Text PDF PubMed Scopus (381) Google Scholar, 16Nishitani H. Nurse P. Cell. 1995; 83: 397-405Abstract Full Text PDF PubMed Scopus (228) Google Scholar). It has been proposed that the cdc18+ gene plays two roles in the cell cycle, mediating the initiation of DNA replication and preventing inappropriate mitosis. In S. cerevisiae, over-expression ofCDC6 induces G2 delay of the cell cycle (17Bueno A. Russell P. EMBO J. 1992; 11: 2167-2176Crossref PubMed Scopus (108) Google Scholar). Both results suggest a possible second function of CDC6 at the G2/M phase boundary. Homologs of yeast Cdc6 have also been found in human (18Williams R.S. Shohet R.V. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 142-147Crossref PubMed Scopus (125) Google Scholar) and Xenopus (19Coleman T.R. Carpenter B.P. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The conservation of Cdc6 among species implies an essential role for this protein. Genomic footprinting has defined two cell cycle stages regarding the formation of replication complexes (RC): pre-RC in G1 phase and post-RC after S phase to M/G1 border (20Diffley J.F.X. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (464) Google Scholar, 21Rowley A. Cocker J.H. Harwood J. Diffley J.F.X. EMBO J. 1995; 14: 2631-2641Crossref PubMed Scopus (161) Google Scholar, 22Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The post-RC footprint closely resembles that produced in vitrowith purified ORC and Abf1, which protect A/B1 and B3 elements, respectively. The pre-RC is defined by its further protecting the B2 element of ARS1 in addition to the A/B1 and B3 elements. The data suggest that the initiation of DNA replication is not controlled by the binding of ORC and Abf1 to the origins; instead, modification of origins has to occur with the involvement of some additional factors. The Cdc6 protein is required for establishment and maintenance of pre-RC, because the arrested cdc6–1 mutant produces a post-replication footprint (23Detweiler C.S. Li J.J. J. Cell Sci. 1997; 110: 753-763PubMed Google Scholar, 24Cocker J.H. Piatti S. Santocanale C. Nasmyth K. Diffley J.F. Nature. 1996; 379: 180-182Crossref PubMed Scopus (291) Google Scholar, 25Santocanale C. Diffley J.F.X. EMBO J. 1996; 15: 6671-6679Crossref PubMed Scopus (142) Google Scholar). The Cdc6 protein is also required for the loading of MCM proteins (26Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 27Donovan 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, 28Aparicio O.M Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 29Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar, 30Perkins G. Diffley J.F.X. Mol. Cell. 1998; 2: 23-32Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, the Cdc6 protein interacts with Cdc28 protein complex after p40sic1 is degraded at late G1 phase (31Elsasser S. Lou F. Wang B. Campbell J.L. Jong A. Mol. Biol. Cell. 1996; 7: 1723-1735Crossref PubMed Scopus (98) Google Scholar). We have recently found that the Cdc6 can stimulate Abf1 binding to the B3 domain of ARS1 DNA fragment (32Feng L. Wang B. Jong A. J. Biol. Chem. 1998; 273: 1298-1302Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Currently, it is believed that interactions of Cdc6 with a variety of other protein complexes, such as the ORC, the MCM complex, and Cdc28 kinase are important in coordinating chromosome replication and cell cycle control (33Dutta A. Bell S.P. Annu. Rev. Cell. Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (339) Google Scholar). However, the detailed molecular mechanism of the CDC6gene action is still largely unknown. There is a conserved nucleotide-binding site in Cdc6 (34Zhou C. Huang S.-H. Jong A.Y. J. Biol. Chem. 1989; 264: 9022-9029Abstract Full Text PDF PubMed Google Scholar). It has been shown that the yeast Cdc6 is able to bind ATP and GTP (10Zwerschke W. Rottjakob H.-W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar). At present, the physiological roles of the nucleotide-binding site are still obscure. One effective way to study the function of the nucleotide-binding site is to carry out site-specific mutagenesis experiments to alter the indispensable Lys residue, and examine its phenotypic consequence. In these studies, we have followed a series of steps to unravel the functions of the nucleotide-binding site. First, we changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We show that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss of function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type. Second, based on the plasmid stability assay and chromosomal DNA synthesis assay, the roles of the conserved Lys is most likely required for DNA replication. This guides us to investigate the formation of a functional replication complex at G1/S transition. Third, we have prepared synchronous chromatin fractions of both mutant and wild-type proteins to examine the effect of the cdc6(K114E) mutation. We found that the loading of the cdc6(K114E) and Mcm5 proteins is drastically altered in the chromatin. Finally, at the molecular level, we demonstrated that Cdc6 interacts with Orc1 in vitro and in vivo. Interestingly, the interaction between Orc1 and Cdc6 is impaired when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP binding of Cdc6. This may lead to assemble the operational replicative complex at the G1/S transition. The biological significance of the ATP binding in this process is discussed in this report. The yeastcdc6–1 strain, 611, has been described (31Elsasser S. Lou F. Wang B. Campbell J.L. Jong A. Mol. Biol. Cell. 1996; 7: 1723-1735Crossref PubMed Scopus (98) Google Scholar). Strain K4055 wasCDC6::hisGURA3hisG,trp1–1::TRP1 MET-CDC6 with URA3 looped out (9Piatti S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (333) Google Scholar). StrainBJ2168 (a prc1–407 prb1–1122 pep4–3 leu2 trpl ura3–52) is a protease-deficient strain. Plasmid carrying alleles mutated at lysine 114 were generated by cloning a 1.8-kbHindIII-EcoRI fragment encompassing theCDC6 ORF into M13mp19, mutagenizing as described below, and cloning the resulting alleles into YEp352 (35Hill J.E. Myers A.M. Koerner T.J. Tzagoloff A. Yeast. 1986; 2: 163-167Crossref PubMed Scopus (1077) Google Scholar). Lysine 114 has been changed to Glu (K114E), Gln (K114Q), Arg (K114R), Pro (K114P), and Leu (K114L) (see Fig. 1). The YEp352 subclones are designated: YEp352-K114E, YEp352-K114L, YEp352-K114R, YEp352-K114P, and YEp352-K114Q (Table I). Plasmid YGp12 for the expression of untagged proteins were prepared by cloning the wild-type and K114E allele under the control of the GAL1,10 promoter in a 2-μm yeast vector with URA3 as selectable marker. Plasmid YGp102 is the same construct except that LEU2 is the selection marker (Fig. 2). YCp5N is derived from a single-copy shuttle vector YCplac111 and contains a 0.7-kb 5′ upstream sequence of the CDC6 gene (34Zhou C. Huang S.-H. Jong A.Y. J. Biol. Chem. 1989; 264: 9022-9029Abstract Full Text PDF PubMed Google Scholar). A T7 tag was inserted into 3′ end of the CDC6 open reading frame in which the TAG codon of CDC6 gene had been removed, and the fusion was subcloned into YCp5N. YEp-HA-Mcm5 is generated by inserting a MCM5 fragment into a high copy vector, YEp352, in which Mcm5 is HA-tagged at its C-terminal 751 amino acids and the expression of the MCM5 gene is driven by its own 444-bp promoter sequence (36Hennessy K.M. Lee A. Chen E. Botstein D. Genes Dev. 1991; 5: 958-969Crossref PubMed Scopus (229) Google Scholar). A full-length of ORC1gene was subcloned into bacterial expression vector pET28b, pET28a-ORC1, for the expression of 6xHis-Orc1 protein YGp181-His-T7-ORC1 contains 6xHis and T7 tag at the N terminus of the ORC1 gene driven by the Galpromoter with Leu2 selection marker (Fig. 7 C). YGp123 is the vector with the insertion of 0.8-kb Gal1-Gal10promoter on the vector YEplac112 with Trp1 as the selection marker. The wild-type CDC6-T7 tag, cdc6(K114E)-T7 tag and temperature-sensitive cdc6–1-T7 tag genes were subcloned into YGp123, individually, for co-expression experiments (Fig. 7 C). Yeast media YPD and CSM medium SD-ura were purchased from Bio101, Inc. All chemicals were ordered from Sigma; and restriction enzymes were purchased from Life Technologies, Inc. or New England Biolabs.Table ITransformation frequency of the wild-type and mutated CDC6 gene into cdc6–1 cells at the permissive (23 °C) and nonpermissive temperature (37 °C)Plasmids23 °C37 °CYEp352-CDC64.8 × 1034.7 × 103YEp352-K114E4.9 × 1031YEp352-K114L4.5 × 1033.5 × 103YEp352-K114R4.2 × 1034.0 × 103YEp352-K114P4.4 × 1032YEp352-K114Q4.7 × 1033.2 × 103Vector only4.0 × 1030Transformations are reported as colonies/ μg of transforming DNA. Open table in a new tab Figure 2Expression of mutant and wild-type Cdc6 proteins in yeast. On the left margin, the protein markers from Life Technologies, Inc. are indicated: myosin (205,000), phosphorylase b (97,400), bovine serum albumin (68,000), ovalbumin (43,000), carbonic anhydrase (29,600), and lysozyme (18,000). CDC6 was expressed under the control of theGAL1,10 promoter as described under “Experimental Procedures.” We created strains carrying either one or two copies of the wild-type or mutant allele by transforming strain BJ2168with YGp12 alone or YGp12 and YGp102 constructs together. Cells were grown in raffinose, and then 2% galactose was added for 3 h; cells were then harvested. Fifty μg of each extract was electrophoresed and blotted, and the blot was probed with polyclonal Cdc6 antibody described previously. Lanes 1 and4, strain BJ2168 carrying YGp12 and YGp102 (vectors only, no CDC6 insert); lane 2, strain BJ2168 carrying YGp12-CDC6;lane 3, strain BJ2168 carrying both YGp12-CDC6 and YGp102-CDC6; lane 5, strain BJ2168 carrying YGp12-K114E;lane 6, strain BJ2168 carrying YGp12-K114E and YGp102-K114E.View Large Image Figure ViewerDownload (PPT)Figure 7Interaction between Orc1 and Cdc6. A, bacterially expressed 6xHis-T7-Orc1 tagged protein was retained on the nickel-chelating matrix used as a protein affinity column (lane 1). The same vector without the Orc1 insert was used as negative control (lane 2). Yeast extracts were prepared from strain BJ5459/YGp123-CDC6 and used as the Cdc6 protein source. The extracts were then passed through the Orc1-charged matrix, washed extensively, and subjected to protein blotting experiments. The retention of Cdc6 protein, if any, was detected by anti-Cdc6 polyclonal antibodies. B, E. coli extracts containing GST-Cdc6 (lane 1), GST-cdc6(K114E) (lane 2), and GST-cdc6–1 (lane 3), individually, were used for Orc1-matrix interaction as described under “Experimental Procedures.” As controls, aliquot of bacterial expressed GST-Cdc6 (lane 4), GST-cdc6(K114E) (lane 5), and GST-cdc6–1 (lane 6) was analyzed by anti-Cdc6 polyclonal antibodies, indicating that they were expressed about the same level.C, yeast strain BJ5459 was transformed with YGp181-His-T7-ORC1 (selection marker Leu2) first. The resulting strain was then transformed separately with either YGp123-Cdc6-T7 (lane 1), or YGp123-K114E-T7 (lane 2), or YGp123-cdc6–1-T7 (lane 3) with selection marker Trp1. 500 ml of culture was induced with 2% galactose for 4 h. The tagged Orc1 was purified by Ni-NTA matrix, concentrated 10-fold, and probed with anti-T7 antibody on the protein blots. The T7 monoclonal antibody can detect not only 6xHis-T7-Orc1 tag protein but also Cdc6-T7, if there is an interaction between Orc1 and Cdc6 proteins. The upper arrow indicates the predicted size of Orc1 and thelower arrow indicates the predicted size of Cdc6.View Large Image Figure ViewerDownload (PPT) Transformations are reported as colonies/ μg of transforming DNA. Muta-Gene in vitromutagenesis kit (Bio-Rad) was used to carry out the experiments as described below. The mutagenic (GGT CCG CCT GGC ACT GGC (G/C)(G/C/T)G ACT) and universal primers were synthesized (12Zhou C. Jong A.Y. DNA Cell Biol. 1993; 12: 363-370Crossref PubMed Scopus (7) Google Scholar, 37Zoller M.J. Smith M. Methods Enzymol. 1983; 100: 468-500Crossref PubMed Scopus (656) Google Scholar); T7 DNA polymerase instead of the Klenow fragment of DNA polymerase I was used in the reaction (38Zhou C. Abaigar L. Jong A. BioTechniques. 1990; 8: 503PubMed Google Scholar). The mutagenized DNA is shown in Fig. 1. Yeast cdc6–1 shows elevated chromosomal loss. The wild-type and mutant cdc6genes were transformed into strain cdc6–1 to examine their plasmid stability. A colony grown on selective medium was resuspended in 0.2 ml of water. A 0.1-ml sample was used to inoculate 5 ml of nonselective media (either YPD or SD plus uracil), and cultures were grown at 30 °C with aeration for 5–10 generations. Dilutions of the initial suspension were plated on YPD plates, and colonies were counted to determine the initial concentration of cells. These plates were then replica-plated to SD-ura to determine the percentage of plasmid-bearing cells. The PFGE labeling method was used to investigate yeast chromosomal DNA synthesis (39Jong A.Y. Wang B. Zhang S.Q. Anal. Biochem. 1995; 227: 32-39Crossref PubMed Scopus (7) Google Scholar). In this method, yeast cells are first labeled with 32Pi in vivo and chromosomal DNA is then resolved by pulsed field gel electrophoresis. Briefly, 20 ml of yeast cell culture was grown at room temperature to the early log phase (∼107 cells/ml) and arrested by α-factor for 90 min. The cells were then washed with 20 ml of low phosphate medium (LPM) three times, and resuspended into the same volume of LPM. After heat treatment (37 °C), 50 μCi of radioactive 32Pi was added. The culture was grown for another 1 h, and uptake was quenched with 50 mm cold phosphate. The labeled cells were harvested and then washed with phosphate-buffered saline buffer, and molecules were separated using the Bio-Rad CHEF-DR II Megabase DNA pulse field electrophoresis system. The resulting gel was dried and autoradiographed. Yeast chromatin was prepared according to Lue and Kornberg (40Lue N.F. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8839-8843Crossref PubMed Scopus (93) Google Scholar) with slight modifications. Briefly, yeast strain K4055 cells harboring plasmid fusions with co-transformation of YEp-Mcm5-HA and YCp5N-Cdc6-T7, or YCp5N-cdc6(K114E)-T7. The transformant cells were grown on the CSM medium in the presence of methionine (MET+) to shut off the endogenous CDC6. The cells were synchronized at G1 with α-factor at final concentration of 10 μg/ml at room temperature for 4 h. The cells were then released from α-factor block, grown on the fresh MET+ medium, and collected at the indicated time points. Aliquots of cells were harvested at different time intervals to monitor the degree of synchrony by measurement of percentage of budded cells and FACS analysis (38Zhou C. Abaigar L. Jong A. BioTechniques. 1990; 8: 503PubMed Google Scholar). Yeast cells were washed once with 50 mm ice-cold EDTA and incubated in a solution containing 20 mm EDTA/2% β-mercaptoethanol at 30 °C for 30 min. After incubation, the cells were centrifuged and washed once with 1m sorbitol. Spheroplasts were generated by digesting the cells with 100 μg/ml yeast lytic enzyme (ICN) in a solution containing 1 m sorbitol, 5 mmβ-mercaptoethanol at 37 °C for 60 min. The spheroplasts were cooled on ice for 10 min, centrifuged at 3000 × g for 10 min at 4 °C, and washed once with the 1 m ice-cold sorbitol. The spheroplasts were collected by centrifugation and lysed in a buffer containing 20% (w/v) Ficoll 400 (Sigma), 20 mmHEPES-KOH, pH 7.5, 20 mm KCl, 5 mmMgCl2, 3 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride (PMSF), and 1 mm EDTA with a Teflon/glass homogenizer. Cell debris were removed by four spins at 3000 × g for 5 min at 4 °C. Uniform white supernatants were recovered and were spun at 36,000 rpm and 4 °C for 30 min. Nuclear pellets were resuspended in 500 μl of the spheroplast lysis buffer described above and subjected to Ficoll 400 gradient centrifugation (20–45%) at 36,000 rpm and 4 °C for 30 min. Chromatin pellets were resuspended in a buffer H/0.1 (50 mmHEPES-KOH, pH 7.5, 100 mm KCl, 5 mm magnesium acetate, 1 mm EDTA, 0.02% Nonidet P-40, 1 mmdithiothreitol, 1 mm PMSF, and 10% glycerol) and stored at −80 °C. 10 μg of protein from each chromatin sample were mixed with 4× SDS loading buffer and boiled in a water bath for 5 min. The samples were resolved by SDS-polyacrylamide gel (8–10%) electrophoresis at 4 °C for 4 h, and proteins were blotted onto an Immobilon-P polyvinylidene difluoride transfer membrane (Millipore). The protein blots were probed either with 12CA5 monoclonal antibody (Boehringer Mannheim) for detecting HA-tagged proteins, or with anti-T7 tag monoclonal antibody (Novagen) for detecting T7-tagged proteins, followed by alkaline phosphatase-conjugated secondary antibody. The protein samples were detected with the Tropix chemiluminescent system. 500 ml of bacterial culture containing expressed 6xHis-T7-Orc1 tagged protein was harvested and resuspended into 15 ml of Buffer I (20 mm Tris-HCl, pH 8, 500 mm NaCl, 20 mm imidazole, 0.1% Triton X-100) in the presence of 20 mm PMSF and 20 mm benzamidine. 10 mg of lysozyme and 2 mg of DNase were added and incubated at 4 °C for 30 min. The mixture was centrifuged at 13,000 rpm for 20 min. The supernatant was collected (∼15 ml) and mixed with 0.2 ml of Ni-NTA (Qiagene) at 4 °C for 60 min. The resulting slurry was packed onto a column and washed with 20 ml of Buffer II (20 mm Tris-HCl, pH 8, 150 mm NaCl, 50 mm imidazole, 0.1% Triton X-100). The Orc1-charged matrix was ready for Cdc6 interaction assay. GST-Cdc6 fusions were prepared from 250 ml of culture. Crude extracts were made in Buffer II with 20 mm PMSF and 20 mm benzamidine. Aliquot was used for SDS-polyacrylamide gel electrophoresis to analyze their expression (Fig. 7 B,lanes 4–6). The rest (∼15 ml) was passed through Orc1-charged matrix 10 times for the interaction studies. For Orc1 and Cdc6 co-precipitation experiments (Fig. 7 C), yeast strain BJ5459 was first transformed with YGp181-His-T7-ORC1 (selection marker Leu2). The resulting strain was then transformed separately with either YGp123-CDC6-T7, or YGp-K114E-T7, or YGp1233-cdc6–1-T7 with selection marker Trp1. 500 ml of culture was induced with 2% galactose for 4 h. The cell pellets were disrupted by Bead-BeaterTM in Buffer II, supplemented with 20 mm benzamidine, 20 mm PMSF, 1 mg of DNase. Tagged Orc1 was purified by Ni-NTA matrix, concentrated 10-fold, and probed with anti-T7 antibody on protein blots. Monoclonal antibody against T7 tag can detect both His-T7-Orc1 and Cdc6-T7 on the same blot if Orc1 can bring down Cdc6-T7 proteins in the experiment shown in Fig.7 C. In vitro site-specific mutagenesis is a powerful tool to probe the relationship between the structure and activity of proteins, because the amino acid residues responsible for a particular function can be identified directly (41Botstein D. Shortle D. Science. 1985; 229: 1193-1201Crossref PubMed Scopus (143) Google Scholar). Previous analysis of the primary sequence (34Zhou C. Huang S.-H. Jong A.Y. J. Biol. Chem. 1989; 264: 9022-9029Abstract Full Text PDF PubMed Google Scholar) has shown that CDC6contains a motif conserved among known ATPases, GXXGXGKT, ranging from residues 108–115 inCDC6 (Fig. 1 A). This element plays a role in binding the pyrophosphate moiety of nucleotides, and the Lys residue is essential for electrostatic interaction between the proteins and the nucleotides (42Moller W. Amons R. FEBS Lett. 1985; 186: 1-7Crossref PubMed Scopus (221) Google Scholar). In order to examine whether the conserved Lys residue represents an essential amino acid residue as in other nucleotide-binding proteins, we have mutated the Lys residue to five other residues. The mutagenic primers were similar to the wild-type sequence except for one or two altered nucleotides at the first two bases in the lysine codon (Fig. 1,A and B). The following substitutions were made at the conserved lysine: to glutamate to test the effect of introducing a negative charge, to proline to test the effect of a general disruption, to glutamine to test the effect of substituting an amide, to leucine to test the effect of introducing a neutral amino acid, and to arginine to test the effect of changing the configuration of the positive charge. Alth" @default.
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• W2017508741 title "The Essential Role of Saccharomyces cerevisiae CDC6 Nucleotide-binding Site in Cell Growth, DNA Synthesis, and Orc1 Association" @default.
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