The Human Homolog of Saccharomyces cerevisiae CDC45

Abstract

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 by the of ORC2L protein in of GST alone expressed from the alone (lane the cDNA probe of CDC45L for on metaphase we mapped the at chromosome 22q11.2 and showed that one of the gene is deleted in patients with DiGeorge syndrome (Fig. This region is deleted in DiGeorge and related and known as DGCR. the entire 2 of sequence in this was deposited in the cDNA sequence of CDC45L with the sequence of we that the gene is the and have the gene The mRNA is from exons over a region in the of the of the are in and of exons are in The in the are nucleotide of the sequences that the A of is nucleotide in a of the are in and of exons are in The in the are nucleotide of the sequences that the A of is nucleotide In budding yeast CDC45 is an essential The and physical interactions of CDC45 protein with ORC and MCM proteins suggest its in the initiation of DNA replication. In cells chromosome origins are at The for initiation of eukaryotic DNA replication is that ORC is to origins In G1 phase other initiation like MCM proteins, and CDC45 with the ORC at origins to form the prereplicative The S cyclin-Cdk and CDC7 kinase the prereplicative complex to DNA replication and also the of initiation complex so that origins cannot be for a time in the same S phase. The homologs of MCM proteins and four of the ORC proteins have been identified in human and other We and have identified human (13Williams R.S. Shohet R.V. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 142-147Crossref PubMed Scopus (125) Google 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). no homolog of CDC45 in eukaryotes has been In this we identified the human homolog of budding yeast The high homology of the cloned cDNA with budding yeast CDC45 and a related protein Tsd2 maydis its as human CDC45L. The mRNA of CDC45L is at G1-S transition, with the in yeast level decreases as cell becomes from 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 decrease in affinity of CDC45L protein for a nuclear tether as S phase proceeds and the physical association with human ORC2L a role of the protein in the initiation of DNA replication. Another is that CDC45L is located at which is deleted in DiGeorge syndrome In one of CDC45L is deleted in patients (Fig. is a of the of and pharyngeal pouches in the is associated with or hypoplasia of and parathyroid and with cardiac abnormalities. The of patients with have deletion in with a similar include Shprintzen which is marked by the and abnormalities, and which has mostly cardiac Several genes have been identified in the DGCR, a putative transcription a receptor for adhesion a and proteins with unknown 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 of a number of genes in the deleted region and in the the that the phenotype may be to than one gene by a CDC45L is the first gene identified in the that is directly required for cell The loss of one may cell in specific during specific or changes in of the of CDC45L may in of the protein produced in specific in and the this we cannot out the that CDC45L is a which is deleted because of its close to other gene whose loss is responsible for the phenotype and that deletion of one of CDC45L has no on cell or on examine the of the of CDC45L in patients and also be at the phenotype of with only one of CDC45L deleted by homologous In we have identified a human homolog of budding yeast CDC45p and U. maydis Tsd2p, which are involved in DNA replication The level of CDC45L increases at G1-S transition 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 a role of the protein in the initiation of mammalian DNA replication. The gene is located in DGCR, and one is deleted in DGS, raising the that this loss may to the phenotype of