Kelly C. Thome

In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G1 and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.

A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situ hybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast.

A new member of the human origin recognition complex (ORC) was cloned and identified as ORC5L. HsORC5p is a 50-kDa protein whose sequence is 38% identical and 62% similar to ORC5p from Drosophila melanogaster. Two alleles of ORC5L were identified, one with and one without an evolutionarily conserved purine nucleotide binding motif. HsORC5p is precipitated from cell extracts with HsORC2p and HsORC4p, indicating that it is part of the putative human ORC. The bulk of HsORC5p is in an insoluble nuclear fraction, whereas the other known human ORC subunits (HsORC1p, HsORC2p, and HsORC4p) are easily extracted in the nuclear-soluble fractions and in S100 (HsORC1p). In addition, we identified an alternatively spliced mRNA from the same locus (HsORC5T). HsORC5Tp also formed a complex with HsORC4p but not with HsORC2p, suggesting it may play a regulatory role in the assembly of different ORC subcomplexes. HsORC5, HsORC5T, and HsORC4 transcripts are abundant in spleen, ovary, and prostate in addition to tissues with high levels of DNA replication like testes and colon mucosa, implicating the human ORC proteins in functions besides DNA replication. Finally, the gene for ORC5L is located at chromosome 7, band q22, in the minimal region deleted in 10% of uterine leiomyomas and in 10-20% of acute myeloid leukemias and myelodysplastic syndromes.

A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue ofSaccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex. A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue ofSaccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex. Initiation of eukaryotic DNA replication involves the controlled and simultaneous firing of numerous sites of initiation. In the budding yeast Saccharomyces cerevisiae, these sites are defined by specific sequences recognized by a multisubunit complex, the origin recognition complex (ORC) 1The abbreviations used are: ORC, origin recognition complex; EST, Expressed Sequence Tag; GST, glutathioneS-transferase. (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (1015) Google Scholar, 2Diffley J.F. Cocker J.H. Nature. 1992; 357: 169-172Crossref PubMed Scopus (297) Google Scholar, 3Bell S.P. Kobayashi R. Stillman B. Science. 1993; 262: 1844-1849Crossref PubMed Scopus (375) Google Scholar, 4Rao H. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2224-2228Crossref PubMed Scopus (177) Google Scholar, 5Rowley A. Cocker J.H. Harwood J. Diffley J.F. EMBO J. 1995; 14: 2631-2641Crossref PubMed Scopus (166) Google Scholar). All six members of ORC identified in yeast are essential for cell viability (3Bell S.P. Kobayashi R. Stillman B. Science. 1993; 262: 1844-1849Crossref PubMed Scopus (375) Google Scholar, 6Bell S.P. Mitchell J. Leber J. Kobayashi R. Stillman B. Cell. 1995; 83: 563-568Abstract Full Text PDF PubMed Scopus (219) Google Scholar, 7Micklem G. Rowley A. Harwood J. Nasmyth K. Diffley J.F. Nature. 1993; 366: 87-89Crossref PubMed Scopus (199) Google Scholar, 8Foss M. McNally F.J. Laurenson P. Rine J. Science. 1993; 262: 1838-1844Crossref PubMed Scopus (270) Google Scholar, 9Hardy C.F. Mol. Cell. Biol. 1996; 16: 1832-1841Crossref PubMed Scopus (60) Google Scholar, 10Loo S. Fox C.A. Rine J. Kobayashi R. Stillman B. Bell S. Mol. Biol. Cell. 1995; 6: 741-756Crossref PubMed Scopus (181) Google Scholar, 11Li J.J. Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (370) Google Scholar). ORC, in its pre-replicative or in its post-replicative form, is bound to DNA throughout the cell-cycle (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (1015) Google Scholar, 2Diffley J.F. Cocker J.H. Nature. 1992; 357: 169-172Crossref PubMed Scopus (297) Google Scholar) and could act as a platform for the recruitment of other proteins involved in the replication machinery. One of the proteins believed to be recruited by ORC before the initiation of DNA replication is the CDC6/Cdc18 (S. cerevisiae or Schizosaccharomyces pombe) protein. CDC6/Cdc18 is closely related in sequence to one of the subunits of ORC, ORC1, over a region that includes a putative nucleotide binding motif. Yeast ORC has been demonstrated to utilize ATP for binding to DNA and to have an ATPase activity that is modulated by binding to the origin of DNA replication (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (1015) Google Scholar, 12Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), suggesting that like DNA replication initiator proteins in Escherichia coli (DnaA) or the simian virus 40 (T antigen), ATP binding and hydrolysis by the eukaryotic initiator protein will be an important regulator of the initiation process. Although DNA sequences defining an origin of replication have not yet been identified in higher eukaryotes, two members of a putative ORC complex homologous to yeast ORC1 and ORC2 have been identified so far in mammals, both in humans and in mice (13Gavin K.A. Hidaka M. Stillman B. Science. 1995; 270: 1667-1671Crossref PubMed Scopus (206) Google Scholar, 14Takahara K. Bong M. Brevard R. Eddy R.L. Haley L.L. Sait S.J. Shows T.B. Hoffman G.G. Greenspan D.S. Genomics. 1996; 31: 119-122Crossref PubMed Scopus (39) Google Scholar), suggesting a universal mechanism of initiation of DNA replication in eukaryotes. We report here the identification of a novel member of human ORC homologous to S. cerevisiae ScORC4. Cloning the gene for a third member of the human origin recognition complex is an important step toward the ultimate goal of reconstituting the entire human ORCin vitro. In the Expressed Sequence Tag (EST) Data base (National Center for Biotechnology Information), the partial sequence of a mouse cDNA (AA168456) was deposited with significant homology to a portion of ScORC4 from S. cerevisiae. A BLAST search with the AA168456 sequence revealed a homologous sequence human EST W23942, which in its turn identified a mouse EST AA110785. A BLAST search with the latter identified a human EST T80329 with an internal portion with significant homology to amino acids 85–121 of ScORC4. T80329 represented the 5′ end of a cDNA clone 25172 (IMAGE, Integrated Molecular Analysis of Genomes and their Expression) obtained from human fetal brain mRNA. This clone was obtained and found to contain a 2.2-kilobase cDNA that corresponds in size to the mRNA detected by Northern blotting. The sequence will be deposited in GenBankTM. A 1-kilobase NcoI fragment from the cDNA encoding amino acids 57–388 of HsORC4P was cloned into the NcoI site of pRSETA. A 35-kDa fragment of HsORC4P was produced in bacteria fused to a 6-histidine epitope tag, purified on a nickel resin column, and used to raise antibodies in rabbits (Cocalico Biologicals). Antibody against human ORC2 was raised against a recombinant His6-tagged fragment of HsORC2P from amino acids 27–577, created by cloning the XbaI-SacI fragment ofHsORC2 cDNA into the PvuII site of pRSETC. Where indicated, antibodies were cross-linked to protein A-Sepharose beads with dimethyl pimelimidate for use in immunoprecipitation experiments; immunoprecipitated proteins were released from the cross-linked antibodies with 100 mm triethylamine, pH 11.5, separated by centrifugation, and brought to Laemmli buffer conditions. To ensure that coimmunoprecipitating proteins were not a result of cross-reacting antibody, interactions were disrupted by boiling the immunoprecipitates in 1% SDS. Samples were then diluted to RIPA buffer conditions and immunoprecipitated again with the same antibodies. We expressed recombinant HsORC4P fused with glutathioneS-transferase (GST) in mammalian cells using the pEBG expression plasmid. Polymerase chain reaction with plaque-forming unit polymerase was used to introduce a BamHI site into theHsORC4 cDNA three nucleotides upstream from the initiator methionine of HsORC4P (GGATCCGAAATG). ThisBamHI site was used to clone HsORC4 cDNA into the pEBG vector such that the GST coding region was fused in-frame to the HsORC4P coding sequence. 293T cells transiently transfected for 48 h with pEBG or pEBG-ORC4 were lysed, and the expressed GST or GST-HsORC4P was recovered by affinity purification on glutathione-agarose beads. Coprecipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting according to standard protocols. Human 293T embryonic kidney cells or HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% donor calf serum. Transfections were done by the standard calcium phosphate method. For metabolic labeling of proteins, 293T cells were incubated in methionine-free medium for 4 h. Then 200 μCi of [35S]methionine (NEN Life Science Products) was added, and cells were incubated for 6 h before harvesting. Cell-staged human cell cultures were prepared from exponentially growing HeLa cells. Cells were arrested in M phase with 40 ng/ml nocodazole for 18 h. Mitotic cells were then selected by shake-off, washed twice in warm PHEM buffer (60 mm PIPES, pH 6.8, 25 mm HEPES, pH 6.8, 10 mm EGTA, 2 mm MgCl2), re-inoculated in plates with fresh medium, and harvested at indicated time points after release. Alternatively, cells were blocked at different cell cycle phases with 10 mm hydroxyurea (S) or 40 ng/ml nocodazole (M) for 18 h. G1 synchronous cells were obtained by nocodazole shake-off as described above and harvested 4 h after re-inoculation. The cell cycle stages of these cells were checked by immunoblotting cell lysates with antibodies to cell cycle-specific cyclin B. Cell extracts were made in 0.1% Nonidet P-40 lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 50 mm NaF, 1 mmNa3VO4) at 4 °C. Where indicated, 200 μg/ml ethidium bromide was added to the buffer to disrupt protein-DNA interactions (15Lai J.S. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (403) Google Scholar). In all cases, the concentration of total protein in the lysates was determined by Bradford assay, and equal amounts were used for immunoprecipitation or for Western blot. Typically, lysate from 107 cells was used for immunoprecipitation, whereas extracts from 5 × 106cells were loaded for Western blots. Total RNA was extracted from HeLa cells as described (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (64946) Google Scholar). 10 μg of RNA/lane was hybridized at 42 °C using a fragment of the HsORC4 cDNA (bases 124–861 encoding the first 245 amino acids) as probe. For reference, the membranes were also hybridized with a 1.3-kilobaseHindIII-PstI cDNA fragment of GAPDH and a cDNA fragment containing the entire open reading frame of cyclin B. Complete sequencing of the cDNA insert revealed an open reading frame encoding a predicted protein of 436 amino acids (Fig.1), with an approximate molecular mass of 45 kDa. The initiator methionine is preceded by an untranslated leader sequence of 126 nucleotides that contains stop codons in all three reading frames. The alignment of the protein sequence reveals 29% sequence identity to ScORC4 (Fig. 1). We have tentatively identified this clone as human replication origin recognition complex ORC4(HsORC4). Amino acids 67–73 of HsORC4 contain a putative NTP binding motif (GXXGXGKT) (17Koonin E.V. Nucleic Acids Res. 1993; 21: 2541-2547Crossref PubMed Scopus (344) Google Scholar). Surprisingly, the sequence of HsORC4 was also significantly related to HsCDC18, SpCdc18, and HsORC1 over areas beyond the nucleotide binding motif (Fig. 2). Nearly 28% of HsORC4 residues were identical to at least one of the three Cdc18/ORC1 sequences shown and have been represented above the alignments as a “consensus sequence.” The identities of HsORC4P with ORC1 and Cdc18 extended over the whole length of the protein, including the six boxes of high homology noted between the Cdc18/ORC1 proteins. Comparing Figs.1 and 2 suggests that ScORC4 contains several of the amino acids that in HsORC4 are related to the Cdc18/ORC1 family. One critical difference, however, is that ScORC4 does not contain the canonical nucleotide binding motif, although ScORC5 has been reported to contain such a sequence motif. Human ORC5 has not yet been identified. It is also clear that the Cdc18/ORC1 molecules are more closely related to each other (especially over the six boxes of homology) than to ORC4 and that the identity of HsORC4P with the HsORC1p or the HsCdc18p is lower (around 17%) than the identity with the budding yeast ORC4P (29%). To study the ORC4 and ORC2 proteins, we raised polyclonal rabbit antibodies to recombinant fragments of the two cloned genes. Fig.3 A shows that immunoprecipitation of cell lysates with the anti-ORC2 antibody (lane 2) isolates a 72-kDa polypeptide that can be detected by immunoblotting with the same antibody. This size is consistent with that reported for HsORC2P. To confirm the specificity of the anti-ORC2 antibody, we expressed Myc epitope-tagged ORC2P by transient transfection of 293T cells with pA3M-ORC2 and immunoprecipitated cell lysates with pre-immune, anti-ORC2, and anti-Myc epitope antibodies (lanes 3–5). The precipitates were immunoblotted with anti-ORC2 antibody. Addition of the Myc epitope to ORC2 produced a longer protein of about 76 kDa, which was precipitated by anti-ORC2 and by anti-Myc antibodies. Thus the anti-ORC2 antibody recognizes ORC2 protein in both immunoprecipitation and immunoblotting reactions. Fig. 3 B shows the specificity of the anti-ORC4 antibody. In this case, endogenous 45-kDa HsORC4 protein was detected by direct immunoblotting of cell lysates (lane 2). In most experiments, a doublet of 45 kDa is seen that could be due to post-translational modifications or to partial proteolysis of HsORC4P. When GST-HsORC4P was expressed in 293T cells by transient transfection of pEBG-ORC4, a new 66-kDa protein was detected by the anti-ORC4 antibody. The size of the new protein and its affinity for glutathione-agarose beads (see below, Fig.4 B) suggests that it is GST-ORC4. This result also proves that the anti-ORC4 antibody recognizes the protein encoded by the cloned HsORC4 cDNA. Lysates from different numbers of HeLa cells were compared with different amounts of purified recombinant HsORC4 by immunoblotting with affinity-purified anti-HsORC4 antibody. Comparison of the relative intensities of signal indicates that approximately 5 × 105 HsORC4 molecules may be present per HeLa cell (data not shown). 293T cells were metabolically labeled with [35S]methionine, and cellular proteins were immunoprecipitated with anti-ORC4 and anti-ORC2 antibodies (Fig.4 A). The anti-ORC4 antibody immunoprecipitated a doublet of 45 kDa and also precipitated polypeptides of 100, 72, 35, and 31 kDa (lanes 2 and 5). With the exception of the 72-kDa band, the intensities of the other three bands are significantly lower than the 45-kDa band. This can be for different reasons. They can be interacting in substoichiometric amounts, they may not be recovered quantitatively, or they can have a slow rate of synthesis. The 45-kDa protein co-migrated with a 45-kDa polypeptide produced by in vitro transcription translation of the HsORC4cDNA (not shown). When the immunoprecipitate was denatured and re-precipitated with anti-ORC4 antibody, only the 45-kDa protein was re-precipitated (lane 3). Taken together with the immunoblotting results in Fig. 3 B, the ORC4 antibody appears to directly recognize only the 45-kDa HsORC4 protein and co-precipitate the other cellular polypeptides because they are associated with HsORC4P. The anti-ORC2 antibody precipitates a protein of an apparent molecular mass of 72 kDa (Fig. 4 A, lane 7), which was the only protein re-immunoprecipitated by the same antibody after denaturation of the proteins (lane 8). Additional polypeptides of 45, 55, 80, and 100 kDa were reproducibly present in the anti-ORC2 immunoprecipitates and are likely to represent proteins associated with HsORC2P. The higher molecular mass polypeptides are better resolved in lane 10. To demonstrate that the 72-kDa protein present in the ORC4 immunoprecipitate was indeed ORC2 and the 45-kDa protein coimmunoprecipitating with ORC2 was ORC4, we repeated the immunoprecipitations with antibodies covalently cross-linked to protein A-Sepharose beads. Bound proteins were eluted, and the presence of the 45-kDa HsORC4P and 72-kDa HsORC2P in the eluates was demonstrated by immunoblotting with the cognate antibodies (Fig. 4 B,lane 2, top and bottom panels). The coimmunoprecipitation experiment was repeated in the presence of 200 μg/ml ethidium bromide (lane 3, top andbottom panels), which is expected to disrupt protein-DNA interactions (15Lai J.S. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (403) Google Scholar). The continued coprecipitation of HsORC4P and HsORC2P in the presence of ethidium bromide supports a direct protein-protein interaction between the two rather than an interaction mediated by a DNA bridge. To ensure that the above results were not due to spurious cross-reaction of the anti-ORC4 antibody with HsORC2P or with a non-ORC4 cellular protein that associates with HsORC2P, we used another method to isolate HsORC4P from cells. Transient transfection of pEBG-ORC4 allowed us to express a 66-kDa GST-HsORC4P fusion protein in 293T cells. In parallel cultures, we expressed GST alone as a negative control from the pEBG vector. GST-HsORC4P (or GST) was isolated from the cell lysates by affinity purification on glutathione-agarose beads (Fig. 4 B, lanes 4 and5, bottom panel). Immunoblotting of the associated proteins with anti-ORC2 antibody revealed that HsORC2P was specifically co-purified with GST-HsORC4 but not with GST alone (Fig.4 B, lanes 4 and 5, top panel). Although HsORC2P and HsORC4P were readily associated with each other in cells, they did not associate with each other when expressed byin vitro transcription translation (data not shown), suggesting that other cellular factors (perhaps other ORC subunits) or post-translational modifications are necessary to mediate stable complex formation. Fig.5 A shows a Northern blot analysis of HsORC4 mRNA expression in asynchronous HeLa cells and cells blocked in M and S phase nocodazole and hydroxyurea respectively. Comparison with the glyceraldehyde-3-phosphate dehydrogenase control indicates that HsORC4 mRNA is essentially unchanged between M and S phases. HeLa cells were also followed as they progressed synchronously after release from an M phase block. HsORC4 mRNA is detected at a constant the cell cycle. The of HsORC4 protein does not through the cell cycle (Fig. 5 Western blot analysis of total cellular protein from cells at indicated time points after release from shows that HsORC4P The cyclin B protein in is shown as a control for a protein expressed in a cell results have been obtained with or cells (not shown). This result is in with the in yeast that levels of ORC remain essentially the cell cycle A. Bell S.P. Cell Biol. 1997; PubMed Scopus (344) Google Scholar). The result is also to the unchanged cellular protein levels of HsORC2P, and HsCdc18p through all phases of the cell cycle. J. A. In we report here the identification of a third member of the human replication origin recognition complex homologous to after which we this gene The homology between human ORC4 and human Cdc18 and S. Cdc18 and human ORC1 suggests that these proteins may be members of a family of related We also that endogenous HsORC4P is associated with endogenous HsORC2P in which in of the of a human ORC we have not yet detected by immunoblotting the anti-ORC4 or anti-ORC2 immunoprecipitates (data not shown). This negative result may be by being recovered in substoichiometric amounts or by the anti-ORC2 or anti-ORC4 antibodies specifically the of ORC1 with the of the proteins and proteins reveals another Although the two proteins are associated with each most of the other proteins present in the immunoprecipitates are HsORC4P is coimmunoprecipitated with proteins of 100, 35, and 31 kDa in to HsORC2P. HsORC2P is coimmunoprecipitated with proteins of 100, 80, and kDa HsORC4P. It is that all these proteins are part of human ORC and have been cell lysis or immunoprecipitation into two Alternatively, the proteins associated with HsORC4P and HsORC2P are not members of human ORC but are other proteins involved in different from the initiation of DNA can be resolved only after the molecular identification of the proteins. In the of has been to be about × on the of the between of about 100 Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). Although the of HsORC4P × is of the two suggests of the two Human cells may contain two of only one of which is in ORC of of DNA Alternatively, all the HsORC4P is in ORC bound to but only a of these sites are as of DNA consistent with the are but are of origin HsORC4 is the third ORC member identified in after HsORC1 and (13Gavin K.A. Hidaka M. Stillman B. Science. 1995; 270: 1667-1671Crossref PubMed Scopus (206) Google Scholar). of ORC in yeast was made by the identification of specific DNA sequence that the in 6Bell S.P. Mitchell J. Leber J. Kobayashi R. Stillman B. Cell. 1995; 83: 563-568Abstract Full Text PDF PubMed Scopus (219) Google Scholar). of sequences in higher has so far We the of human ORC so that it be to in the to human ORC specific DNA sequences and such sequences human The reported in this was the of a to G. by the for on

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. 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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