Xenopus Drf1, a Regulator of Cdc7, Displays Checkpoint-dependent Accumulation on Chromatin during an S-phase Arrest
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m307144200
ISSN1083-351X
AutoresStephanie K. Yanow, Daniel Gold, Hae Yong Yoo, William G. Dunphy,
Tópico(s)Epigenetics and DNA Methylation
ResumoWe have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks. We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts. Drf1 forms an active complex with the kinase Cdc7. However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein. Immunodepletion of Drf1 does not prevent DNA replication in egg extracts. Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels. Nonetheless, Drf1 displays highly regulated binding to replicating chromatin. Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin. This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin. These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin. Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin. This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest. We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts. Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks. In eukaryotes, DNA replication is initiated by a multistep process. Early in the G1-phase, replication initiation factors are sequentially assembled onto replication origins to form prereplicative complexes (pre-RCs). 1The abbreviations used are: pre-RC, pre-replicative complex; XTC, Xenopus tadpole cell; BrdUrd, bromodeoxyuridine; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid; CIB, chromatin isolation buffer; HBS, HEPES-buffered saline.1The abbreviations used are: pre-RC, pre-replicative complex; XTC, Xenopus tadpole cell; BrdUrd, bromodeoxyuridine; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid; CIB, chromatin isolation buffer; HBS, HEPES-buffered saline. At the core of the pre-RC is the origin recognition complex, a six-subunit protein assembly which is required for the subsequent loading of other pre-RC components, including Cdc6/Cdc18, Cdt1, and the Mcms (1Nishitani H. Lygerou Z. 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It has been proposed that the S-phase cyclin-dependent kinases act as global activators of S-phase onset, whereas Dbf4-Cdc7 functions at the level of the individual origins and is therefore required throughout S-phase (11Bousset K. Diffley J.F. Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (239) Google Scholar, 12Donaldson A.D. Fangman W.L. Brewer B.J. Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar). The Dbf4-Cdc7 kinase consists of a regulatory subunit, Dbf4, and a catalytic subunit, Cdc7. In yeast, the Cdc7 subunit is present at constant levels throughout the cell cycle, whereas the Dbf4 subunit accumulates only during the G1/S-phase and is then degraded by ubiquitin-mediated proteolysis (13Pasero P. Duncker B.P. Schwob E. Gasser S.M. Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (101) Google Scholar, 14Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Crossref PubMed Scopus (227) Google Scholar, 15Takeda T. Ogino K. Matsui E. Cho M.K. 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It has been suggested that phosphorylation of Mcm2 alters the structure of the pre-initiation complex to induce DNA unwinding (20Geraghty D.S. Ding M. Heintz N.H. Pederson D.S. J. Biol. Chem. 2000; 275: 18011-18021Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Mcm subcomplexes possess DNA helicase activity in vitro and may be involved in DNA unwinding during origin firing (21Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 22Lee J.K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Crossref PubMed Scopus (158) Google Scholar). In S-phase cells, the presence of stalled replication forks or damaged DNA invokes a checkpoint response that delays entry into mitosis until the defect has been repaired (23Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2633) Google Scholar, 24O'Connell M.J. Walworth N.C. Carr A.M. 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Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1664) Google Scholar). Evidence suggests that ATR is involved in the detection of replication blocks, whereas ATM is activated in response to DNA damage, leading to activation of Chk1 and Chk2, respectively (27Guo Z. Dunphy W.G. Mol. Biol. Cell. 2000; 11: 1535-1546Crossref PubMed Scopus (69) Google Scholar, 28Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 29Hekmat-Nejad M. You Z. Yee M.C. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 30Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar, 31Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (861) Google Scholar, 32Bartek J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Crossref PubMed Scopus (323) Google Scholar). Another protein that has been implicated in the signaling in response to replication blocks is Claspin, which was discovered in Xenopus as a Chk1-binding protein that is essential for activation of Chk1 following induction of the replication checkpoint (33Kumagai A. Dunphy W.G. Mol. Cell. 2000; 6: 839-849Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Recent studies in vertebrate systems have also identified a checkpoint pathway that prevents the onset of DNA replication in the presence of damaged DNA. In Xenopus, one of the targets of this checkpoint pathway is the Cdc45 protein. Reconstitution of the DNA damage checkpoint in a Xenopus cell-free system identified a pathway that activates ATM in response to DNA containing double-strand breaks (7Costanzo V. Robertson K. Ying C.Y. Kim E. Avvedimento E. Gottesman M. Grieco D. Gautier J. Mol. Cell. 2000; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). ATM, in turn, down-regulates cyclin E-Cdk2 activity, thus preventing the loading of Cdc45 onto chromatin and inhibiting DNA replication. Similarly, Cdc45 is a target of a checkpoint induced by treatment with etoposide, a topoisomerase II inhibitor (34Costanzo V. Shechter D. Lupardus P.J. Cimprich K.A. Gottesman M. Gautier J. Mol. Cell. 2003; 11: 203-213Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Activation of this checkpoint, which is mediated by ATR, leads to the down-regulation of Cdc7-associated kinase activity and inhibition of the binding of Cdc45 to chromatin. In yeast, similar pathways are involved in signaling a third checkpoint, the intra-S-phase checkpoint. This checkpoint inhibits the firing of late replication origins in the presence of stalled replication forks or DNA damage that occurs during S-phase. In budding yeast, both subunits of the Dbf4-Cdc7 kinase undergo Rad53-dependent phosphorylation in response to treatment with hydroxyurea. This phosphorylation releases Dbf4-Cdc7 from chromatin, inhibits its kinase activity, and thereby prevents the loading of Cdc45 (4Aparicio O.M. Stout A.M. Bell S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9130-9135Crossref PubMed Scopus (191) Google Scholar, 13Pasero P. Duncker B.P. Schwob E. Gasser S.M. Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (101) Google Scholar, 14Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Crossref PubMed Scopus (227) Google Scholar, 35Kihara M. Nakai W. Asano S. Suzuki A. Kitada K. Kawasaki Y. Johnston L.H. Sugino A. J. Biol. Chem. 2000; 275: 35051-35062Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In mammalian cells, a recent study showed that mice lacking Cdc7 are embryonic lethal, demonstrating that this kinase is essential for embryonic development (36Kim J.M. Nakao K. Nakamura K. Saito I. Katsuki M. Arai K. Masai H. EMBO J. 2002; 21: 2168-2179Crossref PubMed Scopus (71) Google Scholar). Furthermore, when Cdc7 was conditionally removed from an embryonic stem cell line, cells arrested within S-phase with partially replicated DNA. These results support a critical role for the Dbf4-Cdc7 kinase in ensuring the integrity of the DNA throughout the process of replication. In this study we have cloned a Xenopus member of the Dbf4 family, which we have named Xenopus Dbf4-related factor 1 (Drf1). The Drf1 protein is not essential for DNA replication or the recruitment of Cdc45 to chromatin during normal S-phase. However, Drf1 displays highly regulated binding to replicating chromatin. Unlike the scenario in yeast and the etoposide-induced pathway in Xenopus, we have observed that, following replication fork arrest, Drf1 and Cdc7 accumulate on chromatin. This binding is dependent upon ATR and Claspin, and is abrogated by treatment with caffeine. The loss of Drf1 from chromatin in the presence of caffeine correlates with an increase in Cdc45 loading, which is also observed in aphidicolin-treated extracts lacking ATR or Claspin. Caffeine is also capable of overriding an intra-S checkpoint that prevents further initiation events in the presence of aphidicolin, both in XTC cells and in egg extracts. We have established a biochemical assay for this checkpoint by using alkaline agarose gels and have observed a significant increase in the synthesis of small DNA fragments when extracts are treated with aphidicolin and caffeine. We hypothesize that activation of ATR by stalled replication forks leads to the accumulation of Drf1 on chromatin in a checkpoint-dependent manner. This checkpoint-dependent association of Drf1 with chromatin may play a role in preventing the binding of Cdc45 to chromatin during a replication arrest. Cloning of a Xenopus Drf1 Homologue—The following oligonucleotides were designed corresponding to sequences in a Xenopus expressed sequence tag (accession no. BG408573) from the EMBL sequence library: sense, 5′-gcagcaggacgatgaacccccattggcc-3′; antisense, 5′-ccttccgttccgcagcctggatttgggac-3′. A polymerase chain reaction (PCR) using a Xenopus cDNA library generated with the Marathon RACE kit (Clontech) served as the template. The 270-bp PCR product was subsequently biotinylated and used as a probe to screen a Xenopus oocyte cDNA library in the pAX-NMT vector (37Mueller P.R. Coleman T.R. Dunphy W.G. Mol. Biol. Cell. 1995; 6: 119-134Crossref PubMed Scopus (269) Google Scholar) using the ClonCapture cDNA selection kit (Clontech). The same PCR fragment was radiolabeled and used to isolate the full-length cDNA from the pool of clones enriched for Drf1. Positive clones were verified by PCR, and sequencing of both strands was performed with an ABI model 373 automated sequencer. The GenBank™ accession no. for Drf1 is AY328889. Antibodies—Xenopus Cdc7 was amplified by PCR with the following oligonucleotides: 5′-gggaattccatatgagttcgggcgataattcagg-3′ and 5′-actggggaattcctaccgcatgtttttaaacagagc-3′ (38Roberts B.T. Ying C.Y. Gautier J. Maller J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2800-2804Crossref PubMed Scopus (38) Google Scholar). The RACE Xenopus cDNA library described above served as the template. The full-length Cdc7 coding sequence was cloned into the NdeI and EcoRI sites of the pET3-His6X vector, and the resulting plasmid was transformed into Codon Plus Escherichia coli cells for expression and purification as described (38Roberts B.T. Ying C.Y. Gautier J. Maller J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2800-2804Crossref PubMed Scopus (38) Google Scholar). Antibodies were affinity-purified using standard methods with the antigen described above conjugated to CNBr-activated Sepharose 4B (Amersham Biosciences). Antibodies were raised against an N-terminal fragment of Drf1. The fragment was amplified by PCR using the following oligonucleotides: sense, 5′-gggaattccatatgcagcaggacgatgaacc-3′; antisense, 5′-gccaggtgaattcctatgtggggctcac-3′. The 1270-bp fragment was cloned into the NdeI and EcoRI sites of the pET3-His6X vector and expressed in Codon Plus cells. The fusion protein was purified from inclusion bodies as described above for Cdc7. Antibodies were affinity-purified as described above. Affinity-purified antibodies against Xenopus Claspin, Chk1, and ATR, and antisera against Orc2 were described previously (33Kumagai A. Dunphy W.G. Mol. Cell. 2000; 6: 839-849Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 39Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 40Carpenter P.B. Mueller P.R. Dunphy W.G. Nature. 1996; 379: 357-360Crossref PubMed Scopus (175) Google Scholar). Antisera against Xenopus Cdc45 were generously provided by J. Lee. Monoclonal antibodies against human Mcm2 were obtained commercially (BM28, BD Biosciences). Antibodies recognizing Xenopus Mcm4 were kindly provided by J. Blow. Control rabbit IgG was obtained from Zymed Laboratories Inc.. Xenopus GST-Mcm2 was cloned into pGEX4T2 vector (Amersham Biosciences), expressed in Codon Plus cells, and purified with GST-agarose. Recombinant geminin was purified as described (33Kumagai A. Dunphy W.G. Mol. Cell. 2000; 6: 839-849Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 39Lee J. Kumagai A. Dunphy W.G. Mol. Cell. 2003; 11: 329-340Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 40Carpenter P.B. Mueller P.R. Dunphy W.G. Nature. 1996; 379: 357-360Crossref PubMed Scopus (175) Google Scholar). Egg Extracts—Xenopus egg extracts were prepared as described (41Murray A.W. Methods Cell Biol. 1991; 36: 581-605Crossref PubMed Scopus (804) Google Scholar). Egg extracts arrested in interphase because of the presence of unreplicated DNA routinely contained 3000 demembranated Xenopus sperm nuclei/μl of extract and 100 μg/ml aphidicolin. Caffeine was added to a final concentration of 5 mm from a 100 mm solution freshly dissolved in 10 mm Pipes-KOH (pH 7.5). Immunodepletion—Immunodepletions of Chk1 and Claspin from egg extracts were carried out with Affiprep-protein A beads (Bio-Rad) as described previously (33Kumagai A. Dunphy W.G. Mol. Cell. 2000; 6: 839-849Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). For immunodepletion of Cdc7 and Drf1, 100 μl of serum coupled to Affiprep-protein A beads was used per 100 μl of CSF-arrested extract, during two rounds of depletion. Pre-immune serum served as a control. For immunodepletion of ATR, 25 μg of antibody coated on protein A-magnetic beads (Dynal) was incubated with extracts on ice for 1 h. The beads were removed with a magnet, and the procedure was repeated. Chromatin Isolation—Egg extracts (100 μl) containing 3000 sperm nuclei/μl were overlaid on a 1-ml sucrose cushion containing chromatin isolation buffer (CIB; 20 mm HEPES-KOH (pH 7.6), 1 m sucrose, 80 mm KCl, 25 mm potassium gluconate, and 10 mm magnesium gluconate), and centrifuged at 6100 × g for 5 min. The pellets were washed twice with CIB + 0.5% Nonidet P-40, and centrifuged as above. The chromatin pellets were boiled in 2× SDS sample buffer and subjected to SDS-PAGE. To elute chromatin-associated proteins, chromatin pellets were prepared from 400 μl of extract by overlaying 200 μl on a 1-ml sucrose cushion in duplicate. Samples were centrifuged at 6100 × g for 5 min. Chromatin pellets were washed twice with CIB and then once with CIB + 0.5% Nonidet P-40. The supernatants were removed, and the chromatin pellets were resuspended in 100 μl of 1 m NaCl, 10 mm HEPES-KOH (pH 7.6). Samples were incubated on ice for 10 min and then centrifuged at 11,700 × g for 5 min. The supernatant contained the proteins eluted from chromatin. Replication Assays—To monitor DNA replication, egg extracts were incubated with sperm nuclei (1000–3000 sperm nuclei/μl), 0.4 mm CaCl2, 100 μg/ml cycloheximide, and 1 μl of [α-32P]dATP for 90 min at room temperature. The reaction was terminated upon addition of an equal volume of 2× replication stop buffer (80 mm Tris-HCl (pH 8), 8 mm EDTA, 0.13% phosphoric acid, 10% Ficoll, 5% SDS, and 0.2% bromphenol blue), and 1 mg/ml proteinase K, followed by incubation at 55 °C for 1 h. Samples were run on 1% agarose gels, dried, and detected by a PhosphorImager (Amersham Biosciences). Immunoprecipitations and Kinase Assays—For immunoprecipitation of Drf1 and Cdc7 from egg extracts, 100 μl of extract was diluted 3-fold with 10 mm HEPES-KOH (pH 7.6), 150 mm NaCl, 2.5 mm EGTA, 20 mm β-glycerophosphate, and 0.5% Nonidet P-40, and centrifuged at 11,700 × g for 10 min to pellet nuclei. Supernatants were removed and incubated with 5 μg of affinity-purified antibody bound to Affiprep-protein A beads for 1 h at 4 °C with rotation. The beads were washed three times with buffer X (10 mm HEPES-KOH (pH 7.6), 80 mm NaCl, 2.5 mm EGTA, 20 mm β-glycerolphosphate, and 0.1% Nonidet P-40), then once with HBS (150 mm NaCl, 10 mm HEPES-KOH (pH 7.6)). For immunoprecipitation of proteins eluted from chromatin, the supernatant was removed and diluted 8-fold with 50 mm NaCl, 10 mm HEPES-KOH (pH 7.6) and incubated with 5 μg of antibody bound to Affiprep-protein A beads for 1 h at 4 °C with rotation. The beads were washed three times with buffer X, and then once with HBS. For in vitro kinase assays, the beads were resuspended in HBS. One half was boiled in 2× SDS sample buffer, subjected to SDS-PAGE, and immunoblotted for Drf1 and Cdc7. The other half was incubated with kinase buffer (50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 1 mm dithiothreitol, and 50 μm ATP) containing [γ-32P]ATP and 1 μg of GST-Mcm2. Samples were incubated at room temperature for 15 min with rotation, then boiled in 2× SDS sample buffer, subjected to SDS-PAGE, and detected by a PhosphorImager. Cell Culture and Immunofluorescence—XTC-2 cells were grown on poly-d-lysine-coated coverslips in 61% Leibovitz's (L-15) medium supplemented with 10% fetal calf serum and antibiotics. Caffeine-treated cells were incubated with 5 mm caffeine 90 min prior to aphidicolin treatment, where applicable. The cells were then incubated with 100 μm BrdUrd and 5 μg/ml aphidicolin where indicated. At 5.5 h, cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and subjected to indirect immunofluorescence. For BrdUrd visualization, cells were re-fixed with 0.1% formaldehyde and incubated for 10 min in 2 m HCl and 0.1% Triton X-100 at room temperature. The acid was washed away, and the immunofluorescence procedure was repeated with anti-BrdUrd (Roche) as the primary antibody and Texas Red conjugated anti-mouse IgG (Jackson Laboratories) at a 1:500 dilution as the secondary antibody. The coverslips were washed (with 1 μg/ml Hoechst 33258 in the last wash) and mounted onto glass slides with Vectamount (Vector Laboratories). The samples were imaged with a SpotRT CCD camera (Diagnostic Instruments) and analyzed with Adobe Photoshop. Alkaline Gel Electrophoresis—Replication reactions (typically 40 μl of egg extract) were resuspended in 300 μl of StopN (20 mm Tris-HCl (pH 8), 200 mm NaCl, 5 mm EDTA, and 0.5% SDS) containing 2 μg/ml RNase, and digested for 10 min at 37 °C. Proteinase K (200 μg/ml) was added, and samples were incubated for another 30 min at 37 °C. DNA was extracted twice with phenol:chloroform:isoamyl alcohol (Sigma) and then precipitated with ethanol. Pellets were resuspended in 10 μl of 10 mm EDTA, and then diluted with an equal volume of 2× alkaline loading buffer (100 mm NaOH, 2 mm EDTA, 2.5% Ficoll, and 0.025% bromcresol green). Samples were loaded onto gels containing 1% agarose in 50 mm NaCl and 1 mm EDTA, and run overnight in alkaline gel running buffer (50 mm NaOH, 1 mm EDTA). Gels were fixed with 7% trichloroacetic acid, dried, and autoradiographed. Identification of a cDNA Encoding Xenopus Drf1—A Xenopus homologue from the Dbf4 family was cloned by first amplifying a 270-bp sequence with oligonucleotides designed to recognize a Xenopus expressed sequence tag with homology to Dbf4 proteins in other species. This fragment was then used to screen a Xenopus cDNA library to isolate the full-length open reading frame. Recently, a Xenopus Dbf4 sequence has been entered in the GenBank™ data base (accession no. AB095983). Our cloned protein shares significant sequence homology with this protein (28% identical) but represents a second, distinct Dbf4-like polypeptide (Fig. 1A). In humans, two Dbf4-like proteins have been identified, both of which activate Cdc7 (19Jiang W. McDonald D. Hope T.J. Hunter T. EMBO J. 1999; 18: 5703-5713Crossref PubMed Scopus (170) Google Scholar, 42Montagnoli A. Bosotti R. Villa F. Rialland M. Brotherton D. Mercurio C. Berthelsen J. Santocanale C. EMBO J. 2002; 21: 3171-3181Crossref PubMed Scopus (69) Google Scholar). The cDNA that we have identified in Xenopus encodes a 772-amino acid protein that is homologous to both human proteins (32 and 26% identical to human Drf1 and ASK, respectively) but shares a higher identity with Drf1 (Fig. 1A). In comparison with other vertebrate Dbf4 proteins, Xdrf1 shares 29% identity with the hamster Dbf4 and 30% identity with the mouse Dbf4. Based on the sequence and the experimental evidence presented below, we believe that we have cloned a Dbf4-related factor, and have named this protein Xdrf1 (referred to hereafter as Drf1). Three distinct amino acid motifs have been described for the Dbf4 gene family: motifs N, M, and C (43Masai H. Arai K. Biochem. Biophys. Res. Commun. 2000; 275: 228-232Crossref PubMed Scopus (46) Google Scholar). Motif N shares similarity to a BRCT-like domain and may be functionally important for the replication and DNA damage checkpoints; mutations in this region of the fission yeast homologue, Dfp1/Him1, do not affect the replication functions of the kinase but cause hypersensitivity to drugs that block DNA replication or damage DNA (44Ogino K. Takeda T. Matsui E. Iiyama H. Taniyama C. Arai K. Masai H. J. Biol. Chem. 2001; 276: 31376-31387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Motif M consists of a proline-rich region, and motif C resembles a CCHH-type zinc finger motif (43Masai H. Arai K. Biochem. Biophys. Res. Commun. 2000; 275: 228-232Crossref PubMed Scopus (46) Google Scholar). These two regions of Dfp1/Him1 are both necessary and sufficient for full activation of the kinase (44Ogino K. Takeda T. Matsui E. Iiyama H. Taniyama C. Arai K. Masai H. J. Biol. Chem. 2001; 276: 31376-31387Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). All three of these motifs are highly conserved in the Xenopus Drf1 protein (Fig. 1, B and C). Drf1 Is Dispensable for DNA Replication and Cdc45 Loading—We raised antibodies against an N-terminal fragment of the Xenopus Drf1 protein. This antibody efficiently detects the endogenous Drf1 protein by immunoblotting (Fig. 2A). Although the predicted molecular mass of Drf1 is 85 kDa, we found that the protein detected by two different antibodies raised against Drf1 migrated more slowly, at ∼150 kDa. Upon removal of Drf1 with anti-Drf1 antibodies, we observed that a significant amount of Cdc7 remained in the Drf1-depleted extract. Possible explanations are that there is a pool of free Cdc7 or that a second Dbf4-like protein may also form a complex with Cdc7, as is the case in human cells. To determine whether Drf1 is required for DNA synthesis, we depleted Drf1 from egg extracts and monitored the extent of DNA replication. We observed no defect in DNA replication in the absence of Drf1 compared with the mock-depleted or untreated extracts (Fig. 2B). This result contrasts with the nearly complete inhibition of replication that has been reported for depletion of the Xenopus Cdc7 protein (9Jares P. Blow J.J. Genes Dev. 2000; 14: 1528-1540PubMed Google Scholar, 38Roberts B.T. Ying C.Y. Gautier J. Maller J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2800-2804Cr
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