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• W2077047766 abstract "Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G1 to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G1/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G1/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis. Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G1 to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G1/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G1/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis. During successive eukaryotic cell cycles, accurate duplication of the genome is important for the transmission of intact genetic information to daughter cells. The beginning of S phase marks the initiation of chromosomal replication and is therefore strictly regulated by various and complex control systems, necessary for cells to duplicate their genome at the proper time and only once during each cell cycle. Beginning from late M phase to early G1 phase, the origin recognition complex binds to replication origins and recruits Cdc6 and Cdt1 (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar). These two licensing factors collaborate with ORC to load MCM2–7 onto chromatin, which completes assembly of the pre-replicative complex (pre-RC). 4The abbreviations used are: pre-RC, prereplicative complex; pre-IC, preinitiation complex; CDK, cyclin-dependent kinase; DNA pol-ɛ, DNA polymeraseɛ; siRNA, small interfering RNA; RNAi, RNA interference; BrdUrd, bromodeoxyuridine; DAPI, 4′-6′-diamidino-2-phenylindole; RNAi, RNA interference; PCNA, proliferating cell nuclear antigen; pRb, retinoblastoma protein. The onset of S phase in mammalian cells requires the activity of two important kinase complexes, cyclin E/CDK2 and Cdc7/Dbf4 (2Hwang H.C. Clurman B.E. Oncogene. 2005; 24: 2776-2786Crossref PubMed Scopus (364) Google Scholar, 3Masai H. Arai K. J. Cell. Physiol. 2002; 190: 287-296Crossref PubMed Scopus (151) Google Scholar, 4Woo R.A. Poon R.Y. Cell Cycle. 2003; 2: 316-324Crossref PubMed Scopus (175) Google Scholar). These kinases activate the pre-RC, facilitating Cdc45 loading at replication origins and recruitment of DNA polymerases and assembly of the preinitiation complex (pre-IC). Cyclin E/CDK2 kinase complex plays pivotal roles in G1/S transition. It inactivates retinoblastoma protein (pRb) by phosphorylation preventing formation of pRb-chromatin remodeling complexes. As a result, E2F transcription is de-repressed and genes required for DNA replication and S phase progression are up-regulated (5Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (959) Google Scholar, 6Moroy T. Geisen C. Int. J. Biochem. Cell Biol. 2004; 36: 1424-1439Crossref PubMed Scopus (187) Google Scholar). Cyclin E/CDK2 is also believed to be directly implicated in initiation of chromosomal replication by activating replication components via phosphorylation, although its substrates and the activation mechanisms remain to be elucidated (7Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (334) Google Scholar). CDK2 activity is regulated in a cell cycle-dependent manner by its regulatory partner cyclin E, whose protein level is the highest at the G1/S boundary (8Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1051) Google Scholar). It is also negatively regulated by CDK specific inhibitors such as p21 and p27 which bind directly to the cyclinE/CDK2 complex (9Dulic V. Kaufmann W.K. Wilson S.J. Tlsty T.D. Lees E. Harper J.W. Elledge S.J. Reed S.I. Cell. 1994; 76: 1013-1023Abstract Full Text PDF PubMed Scopus (1417) Google Scholar, 10Polyak K. Kato J.Y. Solomon M.J. Sherr C.J. Massague J. Roberts J.M. Koff A. Genes Dev. 1994; 8: 9-22Crossref PubMed Scopus (1834) Google Scholar). Human TopBP1 was first identified as a protein interacting with topoisomerase IIβ (11Yamane K. Kawabata M. Tsuruo T. Eur. J. Biochem. 1997; 250: 794-799Crossref PubMed Scopus (104) Google Scholar). Since TopBP1 contains eight BRCA1 C terminus domains, which are commonly found in proteins involved in DNA repair or cell cycle checkpoints, studies of TopBP1 have mainly focused on its function in the DNA damage response. TopBP1 participates in checkpoint control by responding to various DNA damages (12Greer D.A. Besley B.D. Kennedy K.B. Davey S. Cancer Res. 2003; 63: 4829-4835PubMed Google Scholar, 13Yamane K. Chen J. Kinsella T.J. Cancer Res. 2003; 63: 3049-3053PubMed Google Scholar, 14Yamane K. Wu X. Chen J. Mol. Cell. Biol. 2002; 22: 555-566Crossref PubMed Scopus (153) Google Scholar), and this function is conserved from yeasts to mammals (15Araki H. Leem S.H. Phongdara A. Sugino A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11791-11795Crossref PubMed Scopus (243) Google Scholar, 16Harris S. Kemplen C. Caspari T. Chan C. Lindsay H.D. Poitelea M. Carr A.M. Price C. J. Cell Sci. 2003; 116: 3519-3529Crossref PubMed Scopus (22) Google Scholar, 17Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 18Parrilla-Castellar E.R. Karnitz L.M. J. Biol. Chem. 2003; 278: 45507-45511Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Yamamoto R.R. Axton J.M. Yamamoto Y. Saunders R.D. Glover D.M. Henderson D.S. Genetics. 2000; 156: 711-721Crossref PubMed Google Scholar, 20Yan S. Lindsay H.D. Michael W.M. J. Cell Biol. 2006; 173: 181-186Crossref PubMed Scopus (49) Google Scholar). Additional roles of TopBP1 in transcriptional regulation and chromatin remodeling have been reported: TopBP1 physically interacts with transcriptional factors such as Miz-1 and E2F1 on their target gene promoters and negatively regulates gene transcription (21Herold S. Wanzel M. Beuger V. Frohme C. Beul D. Hillukkala T. Syvaoja J. Saluz H.P. Haenel F. Eilers M. Mol. Cell. 2002; 10: 509-521Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 22Liu K. Luo Y. Lin F.T. Lin W.C. Genes Dev. 2004; 18: 673-686Crossref PubMed Scopus (125) Google Scholar). TopBP1 homologues such as Cut5/Rad4 in Schizosaccharomyces pombe, Dpb11 in Saccharomyces cerevisiae, Mus101 in Drosophila, and Xcut5/Xmus101 in Xenopus are all known to be required for DNA replication and S phase progression in a normal cell cycle as well as DNA damage response. In both fission yeast and Drosophila, their cut5/mus101 mutants show a block in DNA synthesis (19Yamamoto R.R. Axton J.M. Yamamoto Y. Saunders R.D. Glover D.M. Henderson D.S. Genetics. 2000; 156: 711-721Crossref PubMed Google Scholar, 23Saka Y. Yanagida M. Cell. 1993; 74: 383-393Abstract Full Text PDF PubMed Scopus (195) Google Scholar). Dpb11 in budding yeast physically interacts with DNA polymerase ɛ (DNA pol-ɛ) and loaded onto replication origins together in a complex, which is required for recruitment of DNA polymerase α (24Masumoto H. Sugino A. Araki H. Mol. Cell. Biol. 2000; 20: 2809-2817Crossref PubMed Scopus (147) Google Scholar). Xenopus Xcut5, also referred to as Xmus101, is required for recruiting Cdc45 to chromatin (25Hashimoto Y. Takisawa H. EMBO J. 2003; 22: 2526-2535Crossref PubMed Scopus (106) Google Scholar, 26Van Hatten R.A. Tutter A.V. Holway A.H. Khederian A.M. Walter J.C. Michael W.M. J. Cell Biol. 2002; 159: 541-547Crossref PubMed Scopus (107) Google Scholar), which is also true of Cut5 in fission yeast (27Dolan W.P. Sherman D.A. Forsburg S.L. Chromosoma. 2004; 113: 145-156Crossref PubMed Scopus (24) Google Scholar). In human cells, however, the role of TopBP1 in DNA replication or S phase progression has yet to be determined; only the physical interaction between TopBP1 and DNA pol-ɛ implies its involvement in DNA replication (28Makiniemi M. Hillukkala T. Tuusa J. Reini K. Vaara M. Huang D. Pospiech H. Majuri I. Westerling T. Makela T.P. Syvaoja J.E. J. Biol. Chem. 2001; 276: 30399-30406Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In this report, we showed that TopBP1 depletion using small interfering RNAs (siRNAs) induced G1 arrest via down-regulation of cyclin E/CDK2 activity, by increasing levels of p21 and p27. However, although cyclin E/CDK2 kinase activity was restored by co-depletion of p21 and p27 with TopBP1, cells still could not progress into S phase due to a defect in pre-IC formation. Therefore our results suggest a sequential mode of TopBP1 function during G1/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis. Cell Culture and Fluorescence-activated Cell Sorter Analysis—U2OS and Saos-2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Highly confluent cultures promote p27 accumulation, especially in Saos2 cells; therefore, final confluency of all cultured samples was maintained at less than 80%. Cell cycle profiles were analyzed by flow cytometry with standard propidium iodide staining methods. siRNA Transfection—siRNA oligonucleotides (Samchully Pharm Co.) were synthesized to the following target sequences: TopBP1 #1, ACCGUCGUUACACCUUUAG; TopBP1 #2, ACCGAGUACGCCACUCUCA; p21, CCAGCATGACAGATTTCTA; p27, CGACGATTCTTCTACTCAA; control siRNA, CUUACGCUGAGUACUUCGATT (GL3). Cells were transfected with siRNAs at a final concentration of 240 μm using oligofectamine (Invitrogen) according to the instructions of the manufacturer. Transfections were performed up to three times at 24-h intervals for TopBP1 depletion, while one transfection was enough for both p21 and p27 depletion, and this depleted state lasted for 72 h. Thus, for co-depletion with TopBP1, p21 and/or p27 RNAi was performed once at 24 h with the second transfection of TopBP1 siRNA, at which point both p21 and p27 levels have yet to increase in response to TopBP1 depletion. Antibodies and Western Blot Analysis—Anti-TopBP1 polyclonal antibody was raised against the TopBP1 fragment corresponding to amino acids 1019–1167. Anti-Rb and anti-DNA pol-ɛ antibodies (BD Transduction Laboratories), anti-β-actin antibody (Sigma), anti-cyclin D1 antibody (NeoMarkers), anti-MCM2 (Abcam Inc.), and antibodies to phospho-Chk1 (Ser317) and phospho-Chk2 (Thr68) (Cell Signaling) were purchased from the indicated companies. All remaining antibodies were purchased from Santa Cruz Biotechnology. Immunoprecipitation and in Vitro Kinase Assay—Cells were treated with lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 50 mm NaF, 1 mm Na3VO4, and protease inhibitors) for 1 h on a rotator at 4 °C. 100–400 μg of lysates was immunoprecipitated for 1.5 h with anti-cyclin E and -Cdc7 antibodies immobilized on protein G-Sepharose beads (Amersham Biosciences). Kinase assays with the immunoprecipitates were performed as described previously (29Ukomadu C. Dutta A. J. Biol. Chem. 2003; 278: 43586-43594Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 30Sato N. Sato M. Nakayama M. Saitoh R. Arai K. Masai H. Genes Cells. 2003; 8: 451-463Crossref PubMed Scopus (43) Google Scholar). Anti-Cdc7 antibody and GST-MCM2 (carrying N-terminal 60 amino acid residues) used as a substrate for the Cdc7/Dbf4 kinase assay were kind gifts from Dr. Joon-Kyu Lee (Seoul National University). Immunostaining—Cells grown on coverslips were extracted with 0.5% Triton X-100 in CSK buffer (supplemented with 50 mm NaF, 1 mm Na3VO4, and protease inhibitors) for 5 min at 4 °C, followed by 15 min of methanol fixation at -20 °C. For bromodeoxyuridine (BrdUrd) staining, cells on coverslips were incubated with 15 μm BrdUrd (Sigma) for 24 h. After fixing with 4% paraformaldehyde for 15 min at room temperature, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for another 15 min. Anti-BrdUrd antibody (Amersham Biosciences) was supplemented with 1 unit/μl micrococcal nuclease (Worthington) and 20 mm CaCl2 before use. For all immunostaining experiments, primary antibodies were detected with Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories), and coverslips were mounted in Vectashield Mounting Medium (Vector Laboratories) containing 1 μg/ml DAPI. Northern Blotting—Total RNA was extracted from RNAi-treated samples using Trizol (Invitrogen), and 20 μg of each sample was subjected to Northern blotting. Full-length cDNA of p27 or a PCR fragment corresponding to nucleotides 141–492 of the p21 cDNA sequence was labeled with [α-32P]dCTP and used as a probe. Promoter-Luciferase Assay—p21 and p27 promoter regions between -2 kb to +1 were amplified from PAC/BAC clones of RP3–431A14 and RP11–180M15 (BACPAC Resources Center, Children’s Hospital Oakland Research Institute), respectively, followed by cloning into NheI/HindIII site of the pGL3-basic vector (Promega). Cells were transfected with both pCMV-lacZ plasmid and a promoter construct in the ratio of 1:5, which was performed using Lipofectamine PLUS reagent (Invitrogen). Luciferase and β-galactosidase activities were measured 60 h after the transfection. TopBP1-depleted Cells Are Arrested in G1 Phase—To address the role of human TopBP1 during cell cycle progression, we investigated the consequences of loss of TopBP1 function using RNAi method. Six different siRNA oligonucleotides, each targeting a unique sequence of TopBP1 mRNA, were tested for TopBP1 knockdown using GL3 siRNA as a control. U2OS cells were transfected up to three times with the siRNAs at 24-h intervals and harvested at the indicated times (Fig. 1A). TopBP1 protein levels began to decrease after 48 h, and most of TopBP1 was eliminated by 72 h after the first transfection. This depletion lasted for at least 96 h (Fig. 1B). All of the TopBP1 siRNAs efficiently depleted the target protein, and #1 and #2 were used for the remaining experiments (Fig. 1B and supplemental Fig. 1A). Cell cycle profiles of siRNA-treated cells were determined by flow cytometry (Fig. 1C). Compared with the control, TopBP1-depleted cells exhibited an increase in G1 phase cells, while G2/M and S phase populations were relatively decreased. We also observed a considerable increase in the number of floating cells with TopBP1 RNAi. This is consistent with the larger sub-G1 populations present in TopBP1 siRNA-treated cells when we used both floating and attached cells for analyses, suggesting a fraction of TopBP1-deficient cells underwent apoptosis as reported previously (13Yamane K. Chen J. Kinsella T.J. Cancer Res. 2003; 63: 3049-3053PubMed Google Scholar, 22Liu K. Luo Y. Lin F.T. Lin W.C. Genes Dev. 2004; 18: 673-686Crossref PubMed Scopus (125) Google Scholar). Since the sub-G1 populations in both #1 and #2 TopBP1 siRNA-treated cells were not more than 24% until 96 h, we excluded floating cells and focused on non-apoptotic cells in the following experiments. To confirm the cell cycle effects of TopBP1 depletion, cells were synchronized after siRNA transfection using thymidine (Fig. 2, A and B). Following incubation with thymidine, both control and TopBP1-deficient cells were synchronized at the G1/S boundary with 2 n DNA content. However, when cells were released into fresh media, a significant portion of TopBP1-depleted cells retained 2 n DNA content, whereas control cells progressed into S phase. Similarly, a significant population of TopBP1-deficient cells remained in G1 phase in the presence of nocodazole, whereas the control cells arrested in M phase. Therefore our results indicate that TopBP1 depletion causes accumulation of G1-arrested cells. We also obtained similar results using four additional independent TopBP1 siRNAs (supplemental Fig. 1). The levels of cyclins after TopBP1 RNAi were examined by immunoblotting (Fig. 2C). Cyclins E, A, and B are all expressed in a cell-cycle dependent manner, but their expression patterns are distinct from one another; cyclin E protein is maximal at the G1/S transition, while cyclin A and B are expressed during S and M phase, respectively. Cyclin D level is relatively constant throughout the cell cycle, with the exception that it is not expressed in quiescent (G0) cells (31Obaya A.J. Sedivy J.M. Cell Mol. Life Sci. 2002; 59: 126-142Crossref PubMed Scopus (338) Google Scholar). With TopBP1 depletion, no significant changes were observed in the cyclin D levels indicating cells were still in a proliferating state. TopBP1-depleted cells showed an increase in cyclin E accompanied with a decrease in cyclin A and B, even following nocodazole treatment. Considering that cyclin E is degraded in S phase (8Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1051) Google Scholar), these results suggest that TopBP1-deficient cells failed to progress into S phase resulting in the accumulation of cyclin E. The observed decrease in both cyclin A and B with TopBP1 depletion is also consistent with arrest prior to S phase. As a result we narrowed down the cell cycle arrest point of TopBP1 depletion to late G1 phase. To further confirm the cell cycle arrest in G1, DNA replication was measured by BrdUrd incorporation (Fig. 2D). Incubating control cells with BrdUrd for 24 h resulted in the majority of cells (92%) staining positive for BrdUrd incorporation as this was sufficient time for cells to undergo a complete cell cycle. In contrast, BrdUrd incorporation in TopBP1-depleted cells was less than 30% of control cells further supporting the conclusion that TopBP1-deficient cells fail to enter S phase. We also examined the levels of pre-RC subunits and geminin (supplemental Fig. 2). Although most pre-RC subunit levels were not changed by TopBP1 RNAi, the amount of phospho-MCM2 (MCM2-P) decreased significantly, indicating that pre-RC is still inactive in TopBP1-deficient cells (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar, 7Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (334) Google Scholar). On the other hand, geminin was greatly reduced after TopBP1 depletion. This is quite distinct from the double-thymidine block sample (2-Thy) of which geminin level was unchanged (or slightly increased) but Cdt1 has already been degraded. Considered that both geminin accumulation and Cdt1 degradation begin with S phase entry (32McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, 33Zhong W. Feng H. Santiago F.E. Kipreos E.T. Nature. 2003; 423: 885-889Crossref PubMed Scopus (259) Google Scholar), the results are again suggesting that the lack of TopBP1 causes the G1 arrest. Taken together, these data show that TopBP1 depletion results in accumulation of cells with 2 n DNA, increased protein levels of cyclin E, reduced incorporation of BrdUrd, and the low levels of geminin and phospho-MCM2. Therefore, we conclude that cells cannot progress into S phase in the absence of TopBP1 but instead arrest in late G1 phase. The G1 Arrest Caused by TopBP1 Depletion Independent of Both p53 and pRb—p53 and pRb are two well characterized regulators of cell cycle progression. In response to multiple stresses that promote genome instability during the cell cycle, these two proteins induce various signal-transduction pathways, which results in either cell cycle arrest or apoptosis (34Harris S.L. Levine A.J. Oncogene. 2005; 24: 2899-2908Crossref PubMed Scopus (1523) Google Scholar, 35Sherr C.J. McCormick F. Cancer Cell. 2002; 2: 103-112Abstract Full Text Full Text PDF PubMed Scopus (1315) Google Scholar). Since the U2OS cell line in which the G1 arrest occurred upon TopBP1 depletion is both pRb- and p53-positive, we next examined whether the G1 arrest was mediated through either p53 or pRb using different cell lines. Saos-2 is also an osteosarcoma-derived cell line like U2OS but lacks both pRb and p53. When depleted of TopBP1, Saos-2 cells remained in the 2 n state by flow cytometry analyses even when treated with nocodazole or released from a thymidine block. These G1 arrest profiles were even more pronounced than observed in U2OS cells (Fig. 3). Similar results were also obtained using HeLa cells, another pRb- and p53-negative cell line (data not shown). Our results indicate that the G1 arrest following TopBP1 depletion is independent of p53 and pRb. TopBP1 Depletion Reduces Cyclin E/CDK2 Activity by Increasing Levels of p21 and p27—The kinase activity of cyclin E/CDK2 peaks at the G1/S transition and is critical for promoting S phase. Despite having high levels of cyclin E/CDK2 protein, most TopBP1-deficient cells failed to enter S phase. Therefore we were interested in determining whether the activity of cyclin E/CDK2 was affected. We first assessed cyclin E/CDK2 activity by examining the extent of pRb phosphorylation. TopBP1-depleted U2OS cells showed hypophosphorylated pRb in comparison with control cells, indicating reduced cyclin E/CDK2 activity with TopBP1 depletion (Fig. 4A). To measure cyclin E/CDK2 activity directly, in vitro kinase assays were also performed (Fig. 4B). Since cyclin E protein is increased following TopBP1 depletion (Fig. 2C), immunoprecipitation using anti-cyclin E antibody resulted in more immunoprecipitated cyclin E/CDK2 complexes from the extract of TopBP1-deficient cells compared with the control. However, despite the increased cyclin E/CDK2 protein, the kinase activities in TopBP1-depleted U2OS and Saos-2 cells were three to four times lower than the control sample (Fig. 4C). Therefore, we hypothesized that TopBP1 depletion down-regulates cyclin E/CDK2 activity thereby preventing cells from progressing into S phase. The decreased activity also explains the accumulation of cyclin E, since autophosphorylation is required for its degradation via ubiquitin-mediated proteolysis (2Hwang H.C. Clurman B.E. Oncogene. 2005; 24: 2776-2786Crossref PubMed Scopus (364) Google Scholar). To elucidate the mechanism of decreased cyclin E/CDK2 activity in TopBP1-depleted cells, we examined several candidate regulators of CDKs. A number of checkpoint pathways activated by various genomic stresses are reported to target cyclin E/CDK2 and arrest cells in G1 phase, most of which are mediated by Chk1 and Chk2 (36Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (871) Google Scholar, 37Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (651) Google Scholar, 38Uto K. Inoue D. Shimuta K. Nakajo N. Sagata N. EMBO J. 2004; 23: 3386-3396Crossref PubMed Scopus (83) Google Scholar). When TopBP1 was depleted, however, neither phospho-Chk1 nor phospho-Chk2 was detected in both U2OS and Saos-2 cells, whereas hydroxyurea and γ-irradiation treatments demonstrated robust phosphorylation of Chk1 and Chk2. Instead, we observed a dramatic induction of two CDK inhibitors, p21 and p27, in TopBP1-depleted U2OS and Saos-2 cells (Fig. 5A). Time course analyses revealed that the protein levels of p21 and p27 became elevated by 48 h, and this induced state lasted until the 96 h time point (Fig. 5B). Consistent with these results, cyclin E/CDK2 kinase activity began to decrease at 48 h and was further reduced by 96 h. To validate the involvement of p21 and p27 in down-regulation of cyclin E/CDK2 after TopBP1 depletion, the interaction between these CDK inhibitors and cyclin E/CDK2 was examined by immunoprecipitation using anti-cyclin E antibody (Fig. 5C). The amount of co-immunoprecipitated p21 and p27 was significantly increased with TopBP1 depletion compared with that of co-immunoprecipitated CDK2. These results indicate that the up-regulated p21 and p27 indeed interact with cyclin E/CDK2 and inhibit its action in the absence of TopBP1. p21 and p27 Are Induced by Different Mechanisms in the Absence of TopBP1—To investigate the mechanism of p21 and p27 up-regulation following TopBP1 RNAi, we first determined whether p21 and p27 are transcriptionally up-regulated. The mRNA levels of p21 and p27 in siRNA-treated cells were determined by Northern blotting (Fig. 6A). Compared with the control cells, p21 mRNA was greatly induced after TopBP1 depletion in both U2OS and Saos-2 cell lines and correlated well with the temporal induction pattern of the protein. On the other hand, there was no significant change in the p27 mRNA level. Transcriptional activation of p21 and p27 promoters in response to TopBP1 depletion was also examined by performing promoter-luciferase assay (Fig. 6B). Consistent with the Northern blotting results, the luciferase activity of p27 promoter in TopBP1-depleted cells was similar to that in control cells, suggesting non-transcriptional regulation of p27. In contrast, p21 promoter showed a 3-fold increase in luciferase activity with TopBP1 RNAi, confirming that p21 is up-regulated by transcriptional activation in response to TopBP1 depletion. TopBP1 Function Is Required Not Only for Cyclin E/CDK2 Action but Also for Pre-IC Assembly Necessary for S Phase Progression—Last, we determined whether depletion of p21 and/or p27 could suppress the G1 arrest caused by the absence of TopBP1. The p21 and p27 siRNAs efficiently depleted corresponding proteins, but the elimination of either p21 or p27 alone only partially restored cyclin E/CDK2 activity in TopBP1-deficient cells. However, co-depletion of both p21 and p27 recovered about 80% of cyclin E/CDK2 activity (Fig. 7, A and B). The elevated protein levels of cyclin E, shown in TopBP1-depleted cells, also returned to the levels seen in control cells by the co-depletion, demonstrating that kinase activity is required for its autophosphorylation and degradation. Flow cytometry analyses were performed to examine the cell cycle progression of these siRNA-treated cells in the presence or absence of nocodazole (Fig. 7C). When TopBP1 is present, elimination of p21 and/or p27 did not affect cell cycle profiles significantly from control cells. In contrast, TopBP1-deficient cells remained primarily arrested in G1 phase. Depletion of p21 or p27 alone could not rescue this G1 arrest, which is consistent with the in vitro kinase assay results showing only partial restoration of cyclin E/CDK2 activity by depletion of one inhibitor. Thus, it was surprising to find that a large population of cells remained arrested in G1 when both p21 and p27 were depleted with TopBP1 despite restoring cyclin E/CDK2 by 80%. Therefore CDK2 kinase activity is not sufficient for G1/S transition in the absence of TopBP1. These results prompted us to consider Cdc7/Dbf4 kinase, another critical factor for G1/S transition. While cyclin E/CDK2 functions globally for transitioning from G1 to S phase, Cdc7/Dbf4 has been reported to act directly in initiation of DNA replication (39Bousset K. Diffley J.F. Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (239) Google Scholar, 40Donaldson A.D. Fangman W.L. Brewer B.J. Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar). Substantial evidence from yeasts to mammals indicate that MCM proteins are the main substrates of Cdc7/Dbf4 during G1/S transition and that MCM phosphorylation is required for recruiting additional replication components, such as Cdc45 and DNA polymerases, onto replication origins (3Masai H. Arai K. J. Cell. Physiol. 2002; 190: 287-296Crossref PubMed Scopus (151) Google Scholar). Therefore, if Cdc7/Dbf4 activity is also down-regulated by TopBP1 depletion, it could explain how TopBP1/p21/p27 triple-depleted cells remain arrested in G1 phase despite the normal cyclin E/CDK2 activity. To estimate Cdc7/Dbf4 activity, we first examined the phosphorylation of MCM2 protein from siRNA-treated samples (Fig. 8A). Cdc7/Dbf4 phosphorylates a specific residue of MCM2 which increases its mobility on SDS-PAGE (41Montagnoli A. Valsasina B. Brotherton D. Troiani S. Rainoldi S. Tenca P. Molinari A. Santocanale C. J. Biol. Chem. 2006; 281: 10281-10290Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The phospho-MCM2 band (marked with an asterisk) on the SDS-PAGE disappeared in TopBP1-deficient cells, indicating low Cdc7/Dbf4 activity. However, co-depletion of p21 and p27 from TopBP1-depleted cells restored the Cdc7/Dbf4 activity as shown by the normal extent of phospho-MCM2. Furthermore, in vitro kinase assay for Cdc7/Dbf4 activity showed similar results, in which the reduced Cdc7/Dbf4 activity upon TopBP1 depletion was recovered to 60–80% of the control by co-depletion of p21 and p27 (Fig. 8, A and B). Since p21 and p27 are specific inhibitors of CDKs, not of Cdc7, these results suggest that Cdc7/Dbf4 acts after the cyclin E/CDK2 action, which results in the activation of pre-RC by phosphorylating MCM2. To precisely define the point in replication arrested by TopBP1 depletion, next we investigated the loading of two re" @default.
• W2077047766 created "2016-06-24" @default.
• W2077047766 creator A5018058696 @default.
• W2077047766 creator A5024856047 @default.
• W2077047766 creator A5032365993 @default.
• W2077047766 creator A5038170854 @default.
• W2077047766 creator A5042013176 @default.
• W2077047766 creator A5076771980 @default.
• W2077047766 date "2007-05-01" @default.
• W2077047766 modified "2023-10-01" @default.
• W2077047766 title "Human TopBP1 Participates in Cyclin E/CDK2 Activation and Preinitiation Complex Assembly during G1/S Transition" @default.
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