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The Human Homolog of Saccharomyces cerevisiae CDC45

1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês

10.1074/jbc.273.29.18205

1083-351X

Partha Saha, Kelly C. Thome, Ryuji Yamaguchi, Zhi-hui Hou, Stanislawa Weremowicz, Anindya Dutta,

Biofuel production and bioconversion

In budding yeast Saccharomyces cerevisiae CDC45 is an essential gene required for initiation of DNA replication. A structurally related protein Tsd2 is necessary for DNA replication in Ustilago maydis. We have identified and cloned the gene for a human protein homologous to the fungal proteins. The human gene CDC45L is 30 kilobases long and contains 15 introns. The 16 exons encode a protein of 566 amino acids. The human protein is 52 and 49.5% similar to CDC45p and Tsd2p, respectively. The level of CDC45L mRNA peaks at G1-S transition, but total protein amount remains constant throughout the cell cycle. Consistent with a role of CDC45L protein in the initiation of DNA replication it co-immunoprecipitates from cell extracts with a putative replication initiator protein, human ORC2L. In addition, subcellular fractionation indicates that the association of the protein with the nuclear fraction becomes labile as S phase progresses. TheCDC45L gene is located to chromosome 22q11.2 region by cytogenetics and by fluorescence in situ hybridization. This region, known as DiGeorge syndrome critical region, is a minimal area of 2 megabases, which is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by parathyroid hypoplasia, thymic aplasia, or hypoplasia and congenital cardiac abnormalities. CDC45L is the first gene mapped to the DiGeorge syndrome critical region interval whose loss may negatively affect cell proliferation. In budding yeast Saccharomyces cerevisiae CDC45 is an essential gene required for initiation of DNA replication. A structurally related protein Tsd2 is necessary for DNA replication in Ustilago maydis. We have identified and cloned the gene for a human protein homologous to the fungal proteins. The human gene CDC45L is 30 kilobases long and contains 15 introns. The 16 exons encode a protein of 566 amino acids. The human protein is 52 and 49.5% similar to CDC45p and Tsd2p, respectively. The level of CDC45L mRNA peaks at G1-S transition, but total protein amount remains constant throughout the cell cycle. Consistent with a role of CDC45L protein in the initiation of DNA replication it co-immunoprecipitates from cell extracts with a putative replication initiator protein, human ORC2L. In addition, subcellular fractionation indicates that the association of the protein with the nuclear fraction becomes labile as S phase progresses. TheCDC45L gene is located to chromosome 22q11.2 region by cytogenetics and by fluorescence in situ hybridization. This region, known as DiGeorge syndrome critical region, is a minimal area of 2 megabases, which is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by parathyroid hypoplasia, thymic aplasia, or hypoplasia and congenital cardiac abnormalities. CDC45L is the first gene mapped to the DiGeorge syndrome critical region interval whose loss may negatively affect cell proliferation. Eukaryotic DNA replication is regulated during the cell cycle so that it occurs in S phase only once per cycle. This regulation occurs at the level of origin firing. In yeast Saccharomyces cerevisiae, origin recognition complex (ORC) 1The abbreviations used are: ORC, origin recognition complex; DGCR, DiGeorge syndrome critical region;CDC45L, CDC45-like gene in Homo sapiens; FISH, fluorescence in situ hybridization; GST, glutathioneS-transferase; DGS, DiGeorge syndrome. 1The abbreviations used are: ORC, origin recognition complex; DGCR, DiGeorge syndrome critical region;CDC45L, CDC45-like gene in Homo sapiens; FISH, fluorescence in situ hybridization; GST, glutathioneS-transferase; DGS, DiGeorge syndrome. consisting of six subunits (ORC1–6) binds to specific cis-acting DNA sequences(1Bell S.P. Mitchell J. Leber J. Kobayashi R. Stillman B. Cell. 1995; 83: 563-568Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 2Bell S.P. Kobayashi R. Stillman B. Science. 1993; 262: 1844-1849Crossref PubMed Scopus (368) Google Scholar). In human, homologs of four of the ORC subunits (Orc1, Orc2, Orc4, and Orc5) have been identified (3Gavin K.A. Hidaka M. Stillman B. Science. 1995; 270: 1667-1671Crossref PubMed Scopus (204) Google Scholar, 4Quintana D.G. Hou Z.H. Thome K.C. Hendricks M. Saha P. Dutta A. J. Biol. Chem. 1997; 272: 28247-28251Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Homologs of ORC proteins have been identified also in other eukaryotes (5Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar). Although ORC subunits are essential for viability of yeast, their constant binding to replication origins throughout the cell cycle suggests that ORC alone cannot be responsible for the restriction of replication to once per cycle. Another protein, CDC6 in S. cerevisiae (6Zhou C. Huang S.H. Jong A.Y. J. Biol. Chem. 1989; 264: 9022-9029Abstract Full Text PDF PubMed Google Scholar, 7Lisziewicz J. Godany A. Agoston D.V. Kuntzel H. Nucleic Acids Res. 1988; 16: 11507-11520Crossref PubMed Scopus (27) Google Scholar) and Cdc18 inSchizosaccharomyces pombe (8Kelly 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 (384) Google Scholar), is essential for DNA replication and interacts with ORC and cyclin-Cdk. In yeast, CDC6/Cdc18 protein is degraded as the cell cycle progresses through S phase (9Zwerschke W. Rottjakob H.W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar,10Piatti S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (334) Google Scholar). Overexpression of Cdc18 induces re-replication of DNA at S phase in S. pombe (11Nishitani H. Nurse P. Cell. 1995; 83: 397-405Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 12Muzi F.M. Brown G.W. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1566-1570Crossref PubMed Scopus (112) Google Scholar). Homologs of CDC6 have been found in human (13Williams R.S. Shohet R.V. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 142-147Crossref PubMed Scopus (125) Google Scholar, 14Saha P. Chen J. Thome K.C. Lawlis S.J. Hou Z.H. Hendricks M. Parvin J.D. Dutta A. Mol. Cell. Biol. 1998; 18: 2758-2767Crossref PubMed Scopus (217) Google Scholar) and other eukaryotes (15Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Studies with epitope-tagged human CDC6Lp suggest that the human protein is regulated through the cell cycle by changes in subcellular localization (14Saha P. Chen J. Thome K.C. Lawlis S.J. Hou Z.H. Hendricks M. Parvin J.D. Dutta A. Mol. Cell. Biol. 1998; 18: 2758-2767Crossref PubMed Scopus (217) Google Scholar). The epitope-tagged protein is nuclear in G1 and cytoplasmic in S phase. Like Cdc6p, MCM (mini chromosomemaintenance) family of proteins are also implicated in the regulation of initiation of DNA replication. There are six polypeptides in this family (MCM2–7) and homologs identified in human,Drosophila, Xenopus, and S. pombe (5Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar). In yeast, MCM proteins are cytoplasmic except in G1 phase during which the prereplicative complex is formed (16Hennessey K.M. Clark C.D. Botstein D. Genes Dev. 1990; 4: 2252-2263Crossref PubMed Scopus (180) Google Scholar, 17Dalton S. Whitbread L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2514-2518Crossref PubMed Scopus (111) Google Scholar). After mitosis, ORC and Cdc6p recruit MCM proteins to form the prereplicative complex in G1 phase, and DNA replication is initiated upon the activation of the complex by cyclin-Cdk and CDC7 kinases in S phase. CDC45 is yet another gene whose function is required for the initiation of DNA replication in S. cerevisiae (18Hopwood B. Dalton S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12309-12314Crossref PubMed Scopus (92) Google Scholar, 19Zou L. Mitchell J. Stillman B. Mol. Cell. Biol. 1997; 17: 553-563Crossref PubMed Scopus (137) Google Scholar, 20Hardy C. Gene ( Amst. ). 1997; 187: 239-246Crossref PubMed Scopus (48) Google Scholar, 21Owens J.C. Detweiler C.S. Li J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12521-12526Crossref PubMed Scopus (85) Google Scholar).CDC45 genetically interacts with MCM family members and with ORC2 and physically assembles in a complex containing Mcm5p (18Hopwood B. Dalton S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12309-12314Crossref PubMed Scopus (92) Google Scholar, 19Zou L. Mitchell J. Stillman B. Mol. Cell. Biol. 1997; 17: 553-563Crossref PubMed Scopus (137) Google Scholar, 20Hardy C. Gene ( Amst. ). 1997; 187: 239-246Crossref PubMed Scopus (48) Google Scholar). CDC45 protein in yeast is present at a constant level throughout the cell cycle and localized in nucleus (18Hopwood B. Dalton S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12309-12314Crossref PubMed Scopus (92) Google Scholar). CDC45, MCM, and CDC6 proteins together form a complex necessary for the initiation of DNA replication in eukaryotic cells. CDC45 protein is homologous to Tsd2, a protein that is required for DNA replication inUstilago maydis (22Onel K. Holloman W.K. Mol. Gen. Genet. 1997; 253: 463-468Crossref PubMed Scopus (9) Google Scholar). We have identified and cloned a human homolog of yeast CDC45and Tsd2 genes. The CDC45L mRNA level increases during G1-S transition, but the amount of protein is unchanged throughout the cell cycle. Consistent with a role of the protein in the initiation of DNA replication, it is physically associated with human ORC2L protein, and its affinity for a nuclear structure diminishes as DNA replication proceeds during S phase. The gene is located in chromosome 22q11.2 in the minimal region that is deleted in DiGeorge syndrome. DiGeorge syndrome is associated with congenital cardiac abnormalities, hypocalcemia arising from parathyroid hypoplasia, and primary immunodeficiency arising from thymic aplasia. The phenotype may arise from defects in the development of the pharyngeal arches and pouches during embryogenesis (23Morrow B. Goldberg R. Carlson C. Dasgupta R. Sirotkin H. Collins J. Dunham I. Odonnell H. Scambler P. Shprintzen R. Kucherlapati R. Am. J. Hum. Genet. 1995; 56: 1391-1403PubMed Google Scholar, 24Gong W. Emanuel B.S. Collins J. Kim D.H. Wang Z. Chen F. Zhang G. Roe B. Budarf M.L. Hum. Mol. Genet. 1996; 5: 789-800Crossref PubMed Scopus (120) Google Scholar, 25Sirotkin H. Morrow B. Saint J.B. Puech A. Das G.R. Patanjali S.R. Skoultchi A. Weissman S.M. Kucherlapati R. Genomics. 1997; 42: 245-251Crossref PubMed Scopus (91) Google Scholar). Several genes have been identified in the minimal region (2 megabases) commonly deleted. These include a putative transcription factor TUPLE1 (TUP-like enhancer of split gene 1), a potential adhesion receptor protein, a serine threonine kinase DGS-G, and a few genes of unknown function. CDC45L is the first gene consistently deleted in DiGeorge syndrome that may be directly involved in cell proliferation. The EST data base was searched withS. cerevisiae CDC45 nucleotide sequence to look for homologous sequence in human. Two human EST clones had significant matches (T34235 and T31599). The EST clones were obtained from Research Genetics, Inc. Sequencing of the two clones were performed, and the sequences of both clones were the same. The sequence has been deposited in GenBankTM (accession number AF053074) and is the same as another sequence submitted while this manuscript was under review (GenBankTM accession number AJ223728). CDC45L cDNA was cloned into pRSET-C plasmid (Invitrogen) between BamHI and XhoI sites, and the protein was expressed as His6tag in E. coli. The overexpressed protein was purified over nickel-agarose affinity column and used for raising polyclonal antiserum in rabbit (Cocalico Biologicals Inc.). For expressing GST-tagged CDC45L in mammalian cells, pEBG-CDC45L was created by cloning the cDNA into BamHI and KpnI sites of pEBG plasmid (26Saha P. Eichbaum Q. Silberman E.D. Mayer B.J. Dutta A. Mol. Cell. Biol. 1997; 17: 4338-4345Crossref PubMed Scopus (91) Google Scholar). Fluorescence in situhybridization (FISH) was carried out as described (27Ney P.A. Andrews N.C. Jane S.M. Safer B. Purucker M.E. Weremowicz S. Morton C.C. Goff S.C. Orkin S.H. Nienhuis A.W. Mol. Cell. Biol. 1993; 13: 5604-5612Crossref PubMed Scopus (162) Google Scholar) on metaphase chromosome preparations from peripheral blood lymphocytes obtained from normal males and from patients with DiGeorge syndrome known to carry a deletion on one chromosome 21 at the DGCR. HeLa cells were synchronized at mitosis with 50 ng/ml nocodazole (Aldrich) for 24 h. For synchronization in G1-S HeLa cells were blocked with 2 mm thymidine for 12–14 h, released into thymidine-free medium for 12 h, and blocked again with 1 mmhydroxyurea for 12–14 h. The subcellular fractionation was done as described before (28Krude T. Jackman M. Pines J. Laskey R.A. Cell. 1997; 88: 109-119Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). A PstI and XhoI fragment of CDC45L was used to probe the Northern blots. The sequences of the two clones are identical. The 1.86-kilobase cDNA has one long open reading frame, which encodes a protein of 566 amino acids having a theoretical molecular mass of 64 kDa. The polypeptide is highly homologous to CDC45p of S. cerevisiae (27.6% identical and 52% similar) and Tsd2p of U. maydis (26.8% identical and 49.5% similar). As shown in Fig. 1, there is significant homology over the entire length of the human protein with CDC45 and Tsd2. Like the fungal proteins, the human contains a stretch of acidic amino acids (136–166) and a putative bipartite nuclear localization signal (156–172) (Fig. 1). The newly identified protein has no significant sequence homology with any other protein in the data base. Considering its high homology with the yeast CDC45 and Tsd2, we identify the protein as human homolog of the yeast CDC45 (CDC45L). Northern blot analysis of the mRNA from HeLa cells synchronously released from a mitotic block shows that the level of CDC45L mRNA appears at G1-S phase transition (indicated by the increased cyclin E expression and diminished cyclin B expression (29Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 30Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (530) Google Scholar)) and decreases in mitosis (indicated by the increased expression of cyclin B message) (Fig. 2 A). GAPDH mRNA serves as the loading control. The polyclonal antiserum produced against bacterially expressed His6-tagged CDC45Lp specifically recognizes the bacterially expressed antigen and recombinant CDC45Lp expressed in Hi5 insect cells by baculovirus infection (Fig. 2 B). In mammalian cell extract the antiserum recognizes a 60-kDa protein band close to the theoretical size of CDC45L. Western blot analysis of the protein extracts of HeLa cells at various stages of cell cycle shows that although the cells cycle normally (as indicated by the cyclins A and B), the total level of CDC45Lp is unchanged throughout the cell cycle (Fig. 2 C). RPA1 protein is used as a loading control. Subcellular fractionation of asynchronously growing human osteosarcoma U2OS cells indicated that CDC45L protein is present in both the cytosolic and nuclear fractions. Blocking of cells at G1-S phase by aphidicolin (Fig. 2 D, lanes 2 and5) or hydroxyurea (Fig. 2 E, 0 h lane) showed that a significant fraction of the protein was associated with the nuclear fraction. However, blocking of cells in mitosis with nocodazole reveals that the CDC45L protein is now mostly absent from the nuclear fraction (Fig. 2 D, lanes 3 and6). MCM7 protein follows a similar pattern. Thus, like MCM proteins, the affinity of CDC45L protein to a nuclear tether is significantly diminished as DNA replication proceeds. To follow the decrease in nuclear affinity of CDC45Lp during S phase in greater detail, HeLa cells were released from a hydroxyurea block and fractionated at various time points (Fig. 2 E). Pulse labeling with [3H]thymidine indicates that S phase ends at 8 h after release from hydroxyurea block (not shown). Both MCM7p and CDC45Lp are progressively lost from the nuclear fraction as S phase proceeds. Proliferating cell nuclear antigen present in the nuclear fraction was relatively constant at the various time points and serves as a loading and fractionation control. The MCM7p appears to be lost from the nuclear fraction earlier than CDC45Lp, suggesting that the two proteins are released from the nuclear fraction by different mechanisms. The earlier release of human MCMp relative to CDC45Lp agrees well with the recently reported time of release of S. cerevisiae MCM and CDC45 proteins from chromatin (31Zou L. Stillman B. Science. 1998; 280: 593-596Crossref PubMed Scopus (274) Google Scholar). Genetic and physical interactions of yeast CDC45 with yeast ORC2 led us to examine whether the human homologs were physically associated with each other. Because of the co-migration of untagged CDC45L protein with the immunoglobulin heavy chain, evidence for co-precipitation was sought with CDC45L protein tagged at the N terminus with a GST protein. GST-CDC45L (85 kDa) was expressed in 293T cells by transient transfection of EBG-CDC45L. Immunoprecipitation of cell extracts with anti-Orc2 antibody specifically co-precipitated the GST-CDC45L protein (as detected by immunoblotting with anti-GST) (Fig. 2 F, lanes 1 and 2). Conversely, affinity purification of GST-CDC45L from human cell extracts with glutathione agarose beads co-purified human ORC2L protein (lane 4). That this co-purification was because of the CDC45L protein was evidenced by the absence of ORC2L protein in precipitates of GST alone expressed from the EBG vector alone (lane 3). Using the cDNA probe of CDC45L for FISH on metaphase spreads, we mapped the CDC45Lgene at chromosome 22q11.2 and showed that one copy of the gene is deleted in patients with DiGeorge syndrome (Fig. 3). This region is frequently deleted in DiGeorge syndrome, Velocardiofacial syndrome, and related disorders and known as DGCR. Recently the entire 2 megabases of sequence in this DGCR was deposited in GenBankTM. By comparing the cDNA sequence of CDC45L with the genomic sequence of 22q11.2, we confirm that the gene is indeed within the DGCR and have generated the intron-exon boundaries ofCDC45L gene (Table I). The mRNA is transcribed from 19 exons spanned over a 30-kilobase region in the gene.Table ISequences of the exon-intron (HSA) CDC45LSequences of the introns are given in lowercase and those of exons are in uppercase. The numbers in the parentheses are nucleotide coordinates of the exon sequences assuming that the A of ATG is nucleotide 1. Open table in a new tab Sequences of the introns are given in lowercase and those of exons are in uppercase. The numbers in the parentheses are nucleotide coordinates of the exon sequences assuming that the A of ATG is nucleotide 1. In budding yeast CDC45 is an essential gene. The genetic and physical interactions of CDC45 protein with several ORC and MCM proteins suggest its involvement in the initiation of DNA replication. In cdc45-1 cells chromosome origins are fired less frequently at nonpermissive temperature. The overall model for initiation of eukaryotic DNA replication is that ORC is bound to replicator origins constitutively. In G1 phase other initiation factors like CDC6, MCM proteins, and CDC45 associate with the ORC at origins to form the licensed prereplicative complex. The S phase-promoting factors cyclin-Cdk and CDC7 kinase activate the prereplicative complex to initiate DNA replication and also induce the disassembly of initiation complex so that origins cannot be fired for a second time in the same S phase. The homologs of MCM proteins and four of the ORC proteins have been identified in human and other higher eukaryotes. We and others have identified human CDC6L (13Williams R.S. Shohet R.V. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 142-147Crossref PubMed Scopus (125) Google Scholar,14Saha P. Chen J. Thome K.C. Lawlis S.J. Hou Z.H. Hendricks M. Parvin J.D. Dutta A. Mol. Cell. Biol. 1998; 18: 2758-2767Crossref PubMed Scopus (217) Google Scholar). So far no homolog of CDC45 in higher eukaryotes has been reported. In this report, we identified the human homolog of budding yeast CDC45. The high homology of the cloned cDNA with budding yeast CDC45 and a related protein Tsd2 inU. maydis justifies its identification as human CDC45L. The mRNA of CDC45L is highest at G1-S transition, consistent with the observation in yeast where message level decreases as cell population becomes budded from unbudded state (20Hardy C. Gene ( Amst. ). 1997; 187: 239-246Crossref PubMed Scopus (48) Google Scholar). Like the yeast protein, the total protein level remains unchanged during the cell cycle. The selective decrease in affinity of CDC45L protein for a nuclear tether as S phase proceeds and the physical association with human ORC2L support a possible role of the protein in the initiation of DNA replication. Another intriguing observation is that CDC45L is located at Chromosome 22q11.2, which is frequently deleted in DiGeorge syndrome (DGS). In fact, one copy of CDC45L is deleted in DGS patients (Fig. 3). DGS is a developmental anomaly of the derivatives of third and fourth pharyngeal pouches in the embryo. It is associated with aplasia or hypoplasia of thymus and parathyroid glands and with conotruncal cardiac abnormalities. The majority of patients with DGS have deletion in 22q11. Other syndromes with a similar cytogenetic lesion include Shprintzen syndrome, which is marked by the craniofacial and palatal abnormalities, and Takao syndrome, which has mostly cardiac anomalies. Several genes have been identified in the DGCR, including a putative transcription factor, a receptor for adhesion molecule, a serine-threonine kinase, and several proteins with unknown functions (25Sirotkin H. Morrow B. Saint J.B. Puech A. Das G.R. Patanjali S.R. Skoultchi A. Weissman S.M. Kucherlapati R. Genomics. 1997; 42: 245-251Crossref PubMed Scopus (91) Google Scholar). The presence of a number of genes in the common deleted region and variability in the phenotypes raised the possibility that the phenotype may be attributed to more than one gene encompassed by a deletion. CDC45L is the first gene identified in the DGCR that is directly required for cell division. The loss of one copy ofCDC45L may selectively impair cell proliferation in specific tissues during specific developmental stages. Alternatively, mutations, polymorphisms, or changes in methylation status of the remaining allele of CDC45L may result in limiting quantities of the protein being produced in specific tissues resulting in hypoproliferation and the observed developmental anomalies. At this point we cannot rule out the alternative possibility that CDC45L is a bystander gene, which is deleted because of its close proximity to some other gene whose loss is primarily responsible for the DGS phenotype and that deletion of one copy of CDC45L has no effect on cell proliferation or on development. Future experiments will examine the status of the intact allele of CDC45L in DGS patients and will also be directed at determining the phenotype of mice with only one copy of CDC45L selectively deleted by homologous recombination. In summary, we have identified a human homolog of budding yeast CDC45p and U. maydis Tsd2p, which are involved in DNA replication initiation. The RNA level of CDC45L increases at G1-S transition point, but protein level remains constant throughout the cell cycle. However, association of the protein with ORC2L and diminished association with a nuclear tether as S phase proceeds support a role of the protein in the initiation of mammalian DNA replication. The gene is located in DGCR, and one copy ofCDC45L is deleted in DGS, raising the possibility that this loss may contribute to the phenotype of DGS.