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• W2020494268 abstract "Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21Waf1 mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation. Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21Waf1 mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation. Replication of DNA is a strictly regulated event that occurs at a discrete period during the cell cycle. Cell cycle progression is regulated by distinct cyclin-dependent kinases that activate at different times in the cell cycle (reviewed in Ref. 1Reed S.I. Annu. Rev. Cell Biol. 1992; 8: 529-561Crossref PubMed Scopus (266) Google Scholar). In mammalian cells, cyclin E-dependent kinase is activated at the G1 to S phase transition, after the D type cyclins but prior to A type cyclins (reviewed in Ref. 2Pines J. Biochem. Soc. Trans. 1993; 21: 921-925Crossref PubMed Scopus (68) Google Scholar). Microinjection of either anti-cyclin A antibody (3Girard F. Strausfeld U. Fernandez A. Lamb N.J.C. Cell. 1991; 67: 1169-1179Abstract Full Text PDF PubMed Scopus (744) Google Scholar, 4Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1134) Google Scholar) or antisense cyclin A plasmid (3Girard F. Strausfeld U. Fernandez A. Lamb N.J.C. Cell. 1991; 67: 1169-1179Abstract Full Text PDF PubMed Scopus (744) Google Scholar, 5Zindy F. Lamas E. Chenivesse X. Sobczak J. Wang J. Fesquet D. Henglein B. Brechot C. Biochem. Biophys. Res. Commun. 1992; 182: 1144-1154Crossref PubMed Scopus (232) Google Scholar) into exponentially growing cells prevents the entry of cells into S phase. Similarly, microinjection of anti-cyclin E antibody also prevents the entry of cells into S phase (6Ohtsubo M. Theodoras A.M. Schumacher J. Roberts J.M. Pagano M. Mol. Cell. Biol. 1995; 15: 2612-2624Crossref PubMed Scopus (1054) Google Scholar). However, unlike cyclin E-dependent kinase activity, the timing of activation of cyclin A-associated kinase activity in human cells coincides with the onset of DNA synthesis in S phase, which occurs several hours subsequent to the commitment to S phase (7Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (529) Google Scholar, 8Pagano M. Draetta G. Jansen-Durr P. Science. 1992; 255: 1144-1147Crossref PubMed Scopus (178) Google Scholar, 9Rosenblatt J. Gu Y. Morgan D.O. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2824-2828Crossref PubMed Scopus (285) Google Scholar). Cyclin A may therefore have a direct role in DNA replication in S phase. Cyclin-dependent kinases have been implicated as inducers of DNA replication using systems for in vitro replication of DNA. p13suc1, the product of Schizosaccharomyces pombe suc1 gene, binds avidly to active forms of CDC2 and CDK2 (10Labbé J.C. Capony J.P. Caput D. Cavadore J.C. Derancourt J. Kaghad M. Lelias J.M. Picard A. Doree M. EMBO J. 1989; 8: 3053-3058Crossref PubMed Scopus (377) Google Scholar, 11Pondaven P. Meijer L. Beach D. Genes Dev. 1990; 4: 9-17Crossref PubMed Scopus (93) Google Scholar, 12Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (788) Google Scholar). The removal of Cdk2 and Cdc2 proteins from an in vitro Xenopus egg extract replication system, using p13suc1 affinity matrices, has been shown to decrease its ability to replicate sperm DNA (13Blow J.J. Nurse P. Cell. 1990; 62: 855-862Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 14Chevalier S. Tassan J.P. Cox R. Philippe M. Ford C. J. Cell Sci. 1995; 108: 1831-1841PubMed Google Scholar). Specific depletion of Cdk2 protein, but not of Cdc2 protein from Xenopus egg extracts, has been shown to correlate with a decreased ability of these extracts to replicate sperm DNA (15Fang F. Newport J.W. Cell. 1991; 66: 731-742Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Recently, a similar inhibition of DNA replication was observed following depletion of cyclin E from Xenopus egg extracts (16Jackson P.K. Chevalier S. Philippe M. Kirschner M.W. J. Cell Biol. 1995; 130: 755-769Crossref PubMed Scopus (189) Google Scholar). In the SV40 in vitro replication system the inability of human G1 extracts to replicate SV40 origin containing DNA (17Roberts J.M. D'Urso G. Science. 1988; 241: 1486-1489Crossref PubMed Scopus (32) Google Scholar) can be overcome by the addition of cyclin A (18D'Urso G. Marraccino R.L. Marshak D.R. Roberts J.M. Science. 1990; 250: 786-791Crossref PubMed Scopus (201) Google Scholar) or an active CDC2 kinase (19Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (224) Google Scholar). Both cyclin A and Cdk2 are associated with SV40 origin containing DNA during replication in vitro (20Fotedar R. Roberts J.M. Cold Spring Harbor Symp. Quant. Biol. 1991; 56: 325-333Crossref PubMed Scopus (26) Google Scholar). RPA, a cellular single-stranded DNA binding protein, and SV40 T antigen are both phosphorylated by Cdc2-associated kinase in vitro. Phosphorylation of bacterially expressed T antigen by cdc2 kinase increases its affinity for the SV40 origin of replication (21McVey D. Brizuela L. Mohr I. Marshak D.R. Gluzman Y. Beach D. Nature. 1989; 341: 503-507Crossref PubMed Scopus (144) Google Scholar). However, the stimulation of DNA replication by CDC2 kinase in G1 cell extracts was shown not to be due to phosphorylation of T antigen by CDC2 kinase (18D'Urso G. Marraccino R.L. Marshak D.R. Roberts J.M. Science. 1990; 250: 786-791Crossref PubMed Scopus (201) Google Scholar, 19Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (224) Google Scholar). Instead, these studies suggested that phosphorylation of the 34-kDa subunit of RPA during G1 to S phase transition may contribute to the activation of origin unwinding and subsequent DNA replication. The functional significance of phosphorylation of the 34-kDa subunit of RPA by CDC2 kinase (19Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (224) Google Scholar, 22Pan Z.Q. Amin A.A. Gibbs E. Niu H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8343-8347Crossref PubMed Scopus (105) Google Scholar) remains to be established, since mutation of the CDC2 consensus phosphorylation sites on RPA-34 has no effect on in vitro SV40 replication (23Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar). Although previous studies, discussed above, have implicated cdk-cyclins in DNA replication, such studies have not explicitly addressed whether it is the kinase activity itself which is important for DNA replication in S phase or if the cdk-cyclin plays a structural role in replication. Reconstitution of replication activity in G1 extracts by the addition of cyclin A or of Cdc2 kinase demonstrates a role for cdk-cyclins in activating events in G1, but it does not address whether the kinase is required during replication in S phase cell extracts (18D'Urso G. Marraccino R.L. Marshak D.R. Roberts J.M. Science. 1990; 250: 786-791Crossref PubMed Scopus (201) Google Scholar, 19Dutta A. Stillman B. EMBO J. 1992; 11: 2189-2199Crossref PubMed Scopus (224) Google Scholar). Here we have used the SV40 in vitro replication system to examine the regulation of DNA replication by cyclin-associated kinases in S phase cell extracts, and we have specifically addressed whether cyclin-dependent kinase activity is important for DNA replication. Furthermore, since both cyclins A and E are present in S phase cell extracts, we have addressed whether the cyclin A or the cyclin E-dependent kinase has a specific function during DNA replication. We show that cyclin A is specifically required for efficient replication of DNA in S phase, since complete immunodepletion of cyclin A-associated kinase from S phase cell extracts leads to a decrease in the ability of these extracts to support DNA replication. Addition of either purified cyclin A-associated CDK2 kinase or of purified cyclin A alone, but not of cyclin E or cyclin B, fully restores replication activity in these depleted extracts. Furthermore, an increase in kinase activity is specifically required for the reconstitution of replication activity of cyclin A-depleted cell extracts. We have also determined if DNA replication can be suppressed by specific interaction of p21Waf1/Cip1, an inhibitor of cyclin-dependent kinases (24Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar, 25Gu Y. Turck C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (709) Google Scholar, 26Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3179) Google Scholar), with Cdk-cyclin complexes. Since p21 protein also binds to and inhibits PCNA 1The abbreviations used are: PCNAproliferating cell nuclear antigenGSTglutathione S-transferasePCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisRPAreplication factor A. (27Flores-Rozas H. Kelman Z. Dean F.B. Pan Z.Q. Harper J.W. Elledge S.J. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8655-8659Crossref PubMed Scopus (417) Google Scholar, 28Waga S. Hannon G.J. Beach D. Stillman B. Nature. 1994; 369: 574-578Crossref PubMed Scopus (1591) Google Scholar), a protein required for replication, we have used p21 mutant proteins that bind Cdk-cyclin and inhibit both cyclin A and E kinases but do not bind PCNA. These mutant p21 proteins inhibit DNA replication to the same extent as obtained through immunodepletion of cyclin A from S phase cell extracts. Finally, and most importantly, we show that the cyclin-dependent kinase is not required for the formation of initiation complexes but is rate-limiting at a stage before elongation. proliferating cell nuclear antigen glutathione S-transferase polymerase chain reaction polyacrylamide gel electrophoresis replication factor A. Manca cells (a human Burkitt lymphoma line) were grown in spinner flasks with RPMI 1640 containing 5% calf bovine serum and 2 mM L-glutamine (29Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (121) Google Scholar, 30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). Exponentially growing cells were blocked with 2 mM hydroxyurea for 12 h to obtain Manca cells in S phase. Released cells were stained with propidium iodide and analyzed for DNA content by flow cytometric analysis. S-100 supernatants containing 100 mM NaCl were made from hypotonic lysates of Manca cells in S phase as described earlier (29Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (121) Google Scholar, 30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). Replication reactions (31Stillman B.W. Gluzman Y. Mol. Cell. Biol. 1985; 5: 2051-2060Crossref PubMed Scopus (253) Google Scholar) were performed for 90 min at 37°C as described earlier with the exception that 150 ng of SV40 origin containing DNA was used in 50-μl reactions, and the SV40 T antigen (1 μg) used in these experiments was prepared from insect cells (Sf9) infected with baculovirus vector expressing SV40 T antigen (29Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (121) Google Scholar, 30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). Replication reactions were performed in the presence of [α-32P]dCTP, and DNA synthesis was quantitated by measuring the incorporation of [α-32P]dCMP into trichloroacetic acid-precipitable counts (31Stillman B.W. Gluzman Y. Mol. Cell. Biol. 1985; 5: 2051-2060Crossref PubMed Scopus (253) Google Scholar). In all reactions, the protein concentration of S phase cell extracts was adjusted to 100 μg per 50-μl reaction. In Fig. 8, the effect of p21 proteins and the effect of cyclin A addition on DNA elongation was determined by performing replication in two steps. In the first step, SV40 T antigen (1 μg) was preincubated for 30 min at 37°C with S-100 S phase Manca cell extracts (100 μg) and with SV40 origin containing plasmids (150 ng) in the presence of 3 mM ATP to allow the formation of initiation complexes on DNA (29Fotedar R. Roberts J.M. EMBO J. 1992; 11: 2177-2187Crossref PubMed Scopus (121) Google Scholar, 32Wobbe C.R. Dean F.B. Murakami Y. Weissbach L. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4612-4616Crossref PubMed Scopus (28) Google Scholar). In the second step, elongation was initiated by the addition of ribonucleoside triphosphates (except ATP) and deoxyribonucleoside triphosphates. In Fig. 8B, cyclin A was added after 5 min of elongation. In Fig. 8C, after 10 min of elongation, GST-p21, p21 mutant protein or GST control protein were added. The reactions were then allowed to continue for an additional 20 min at 37°C to assay the effect of p21 on elongation. In Fig. 9B, cyclin A was added at the time of elongation.Fig. 9Cyclin-dependent kinase activity is required to activate assembled initiation complexes. A, replication reactions were performed with 150 ng of SV40 origin containing DNA and 100 μg of control cell extracts. DNA synthesis was then measured at the indicated times. A lag of 15 min precedes the start of DNA synthesis. B, replication reactions were performed in two steps. GST-cyclin A was added to reactions containing cyclin A-depleted S phase extracts after initiation, at the onset of elongation (filled squares). DNA synthesis was then measured at the indicated times during the elongation phase. The data shown represent the means for three separate experiments. Standard deviation of the data shown do not exceed 10%. Elongation in cyclin A-depleted extracts, treated identically, is shown for comparison (open squares).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Fig. 10, the rate of DNA elongation was determined by performing replication in two steps as described above but with the following modifications for pulse-chase analysis (Fig. 10C). Briefly, in the first step, SV40 T antigen was preincubated for 20 min at 37°C with either cyclin A-depleted or control cell extracts and with SV40 origin containing plasmids in the presence of 3 mM ATP to allow the formation of initiation complexes on DNA. In the second step, elongation was initiated by the addition of the remaining ribonucleoside triphosphates, dTTP and dATP. At this time the reaction was given a pulse of [α-32P]dCTP for 30 s (33Eki T. Matsumoto T. Murakami Y. Hurwitz J. J. Biol. Chem. 1992; 267: 7284-7294Abstract Full Text PDF PubMed Google Scholar). The elongation reaction was limited during the pulse by the absence of dGTP. The pulse was terminated by adding 100-fold excess cold dCTP, and dGTP was added to the reaction to allow elongation during the chase. The reaction was then allowed to continue at 37°C. Aliquots were removed at various time points during the chase, and replication was terminated at each time point by adding an equal volume of 0.4% SDS and 100 mM EDTA. The reaction products were then digested with proteinase K at 37°C for 1 h. Finally, the samples were extracted with equal volume of chloroform:phenol, precipitated with ethanol, and resuspended in 50 mM NaOH, 1 mM EDTA, and then analyzed on a 1.2% agarose gel in 30 mM NaOH, 1 mM EDTA (34Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19723-19733Abstract Full Text PDF PubMed Google Scholar). After electrophoresis, the agarose gel was fixed in 10% trichloroacetic acid for 20 min followed by 15 min in 10% acetic acid, 12% methanol. The gel was then rinsed with distilled water, dried on DE81 paper (Whatmann), and autoradiographed. Rabbit polyclonal antisera, raised against bacterially expressed human cyclin A and affinity purified antibodies, were kindly provided to us by M. Ohtsubo and J. Roberts (Fred Hutchinson Cancer Research Center, Seattle). The specificity of anti-cyclin A antibody has been confirmed using recombinant cyclin proteins. Specificity of cyclin A antibodies was additionally confirmed by immunoprecipitating cyclin A from G1, S, and mitotic phase extracts and assaying for histone H-1 kinase activity (30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). Rabbit polyclonal antiserum was also raised against synthetic peptide corresponding to the C-terminal sequences of human CDK2 (CDVTKPVPHLRL) (30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). Antiserum to CDK2 was affinity purified on a Sepharose 4B column containing covalently coupled peptide. Monoclonal antibody recognizing human PCNA was purchased from Coulter Immunology (Clone 19A2) and monoclonal antibody recognizing cyclin E was purchased from Oncogene. All the steps of immunodepletion were performed at 4°C. S phase Manca cell extract (25 mg of S-100 cell extract) was adjusted to 40 mM Hepes (pH 7.5), 8 mM MgCl2, 100 mM NaCl, 0.5% Nonidet P-40, 1 μg/ml each aprotinin and leupeptin (IP buffer) and loaded on a column containing 1 ml of packed protein A-Sepharose (Sigma) coupled to anti-cyclin A antibody. Extracts passed over Protein A-Sepharose coupled with nonimmune rabbit immunoglobulins were similarly treated and used as controls. After 20 passages through the column, the flow-through comprising the depleted cell extract was collected, aliquoted, and stored at −70°C for further use in the replication assay. The columns were regenerated with 100 mM glycine HCl (pH 3.0) after use, washed with IP buffer without Nonidet P-40, and stored in the presence of 0.02% sodium azide. All the steps were performed at 4°C, and the tubes were gently rocked during the incubations. 100 μg of S phase cell extract was adjusted to 40 mM Hepes, 8 mM MgCl2, 100 mM NaCl, 0.5% Nonidet P-40, 1 μg/ml each aprotinin and leupeptin (IP buffer) and added to 10 μl of packed Protein A-Sepharose (Sigma) that had been preincubated with anti-cyclin A antibody for 1 h and washed five times with IP buffer. After 1 h, Protein A-Sepharose containing the cyclin A immunocomplexes was washed three times with IP buffer and twice with kinase buffer (40 mM Hepes, 8 mM MgCl2). Kinase assays were performed in 18 μl of kinase reaction mixture containing 40 mM Hepes, 8 mM MgCl2, 166 μM ATP, 5 μCi of [γ-32P]ATP (DuPont NEN, 3,000 Ci/mmol), 4 μg of histone H-1 (Boehringer Mannheim), and 10 μl of packed Protein A-Sepharose as described earlier (30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). After 20 min at 37°C, the reactions were stopped by adding SDS sample buffer. Two-fifths of the reactions were loaded on 12% SDS-polyacrylamide gels. The gels were stained with Coomassie Brilliant Blue, dried, and autoradiographed. For quantitation, the histone H-1 bands were excised from the gel and subjected to scintillation counting. Proteins were resolved on 12% SDS-polyacrylamide gels and immunoblotted as described earlier (30Brenot-Bosc F. Gupta S. Margolis R.L. Fotedar R. Chromosoma (Berl.). 1995; 103: 517-527Crossref PubMed Scopus (52) Google Scholar). The nitrocellulose filters were processed for ECL (enhanced chemiluminescence system; Pharmacia Biotech Inc.) Western blot procedure as instructed by the suppliers. The full-length p21 cDNA was generated by reverse transcription of Jurkat poly(A)+ RNA, followed by polymerase chain reaction (PCR) using two sets of nested p21-specific primers. Sequence analysis revealed that the PCR product was identical in sequence to the published human p21 cDNA (35El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar). The deletion mutants of p21 were generated by polymerase chain reaction (PCR), and the full-length p21 and PCR products of mutant p21 were cloned in-frame into a pGEX vector that expressed them as GST-fusion proteins. The recombinant protein was produced in Escherichia coli (DH5α) as described earlier (36Fotedar R. Fitzgerald P. Rousselle T. Cannella D. Doree M. Messier H. Fotedar A. Oncogene. 1996; 12: 2155-2164PubMed Google Scholar). Cyclin B-Cdc2 kinase was purified to apparent homogeneity from maturing starfish oocytes as described previously (37Labbé J.C. Cavadore J.C. Doree M. Methods Enzymol. 1991; 200: 291-301Crossref PubMed Scopus (41) Google Scholar). Recombinant cyclin A-CDK2 kinase was prepared from human cyclin A and GST-CDK2, both produced in E. coli and purified as described in Lorca et al. (38Lorca T. Labbé J.C. Devault A. Fesquet D. Strausfeld U. Nilsson J. Nygren P.A. Uhlen M. Cavadore J.C. Doree M. J. Cell Sci. 1992; 102: 55-62PubMed Google Scholar). Highly purified CAK (Cdk Activating Kinase), purified to the Mono S step from starfish oocytes (39Fesquet D. Labbé J.C. Derancourt J. Capony J.P. Galas S. Girard F. Lorca T. Shuttleworth J. Doree M. Cavadore J.C. EMBO J. 1993; 12: 3111-3121Crossref PubMed Scopus (327) Google Scholar), was used to stoichiometrically phosphorylate GST-cdk2 in a mixture containing 0.33 mM ATP, 16.6 μM MgCl2, 30 μg/ml GST-cdk2, and 100 μg/ml cyclin A in 10 mM Tris (pH 7.5). The GST-CDK2-cyclin A kinase was then purified using glutathione affinity matrix by standard procedures. GST-cyclin A (40Atherton-Fessler S. Parker L.L. Geahlen R.L. Piwnica-Worms H. Mol. Cell. Biol. 1993; 13: 1675-1685Crossref PubMed Scopus (151) Google Scholar), GST-cyclin B (41Solomon M.J. Glotzer M. Lee T.H. Philippe M. Kirschner M.W. Cell. 1990; 63: 1013-1024Abstract Full Text PDF PubMed Scopus (506) Google Scholar), and GST-cyclin E (42Koff A. Cross F. Fisher A. Schumacher J. Leguellec K. Philippe M. Roberts J.M. Cell. 1991; 66: 1217-1228Abstract Full Text PDF PubMed Scopus (515) Google Scholar) were produced in bacteria and purified by identical procedures using a glutathione affinity matrix as described (36Fotedar R. Fitzgerald P. Rousselle T. Cannella D. Doree M. Messier H. Fotedar A. Oncogene. 1996; 12: 2155-2164PubMed Google Scholar). Cyclin A was immunodepleted from S phase cell extracts using a cyclin A-specific antibody, under conditions that were compatible with subsequent use of the depleted cell extract for replication assay, as described under “Experimental Procedures.” Following depletion, there was no detectable cyclin A in the depleted extract (Fig. 1). Control S phase extracts treated identically with nonimmune rabbit antibody completely retain cyclin A (Fig. 1). Cyclin A has been reported to be in a quaternary complex with proliferating nuclear cell antigen (PCNA), p21, and Cdk2 in nontransformed cells (43Xiong Y. Zhang H. Beach D. Genes Dev. 1993; 7: 1572-1583Crossref PubMed Scopus (486) Google Scholar). We therefore determined if depletion of cyclin A resulted in a loss of PCNA that could consequently affect the replication activity of the extracts. Fig. 1 shows that the level of PCNA was not significantly different following depletion of cyclin A from S phase cell extracts, in comparison to control extracts (Fig. 1). Depletion of cyclin A from S phase extracts results in complete loss of cyclin A-associated H-1 kinase activity (Fig. 1), although the amount of CDK2 protein does not change significantly after depletion (Fig. 1). There is a substantial loss of CDK2-associated kinase activity in cyclin A-depleted extracts (Fig. 1). This result suggests that the active form of CDK2 in S phase extracts is predominantly associated with cyclin A and constitutes a small fraction of the total CDK2. In keeping with this result, the kinase activity associated with cyclin E in these cell extracts was 10-fold lower than that associated with cyclin A or CDK2 (data not shown). DNA replication in control extracts treated with nonimmune rabbit antibody is largely completed by 90 min (data not shown). The extent of DNA synthesis in cyclin A-depleted S phase cell extracts was therefore compared with control extracts after 90 min of reaction. In four independent experiments, cyclin A-depleted extracts exhibited 40-60% of the level of replication observed in control extracts (Fig. 2). It is noteworthy that the inhibition of DNA replication observed in cyclin A-depleted extracts cannot be prevented by the presence of phosphatase inhibitors in depleted cell extracts, indicating that the inhibition is not due to dephosphorylation of previously phosphorylated proteins required for replication. Complete depletion of cyclin A from S phase extracts did not lead to total inhibition of DNA replication. To test if cyclin E kinase activity may be responsible for activating the residual levels of DNA replication, cyclin E was immunodepleted from S phase extracts. When these extracts were tested for replication activity, there was only a 10-15% reduction of DNA replication compared with control extracts. Similarly, immunodepletion of both cyclin A and cyclin E from S phase extracts results in only an additional 10-15% inhibition of DNA replication, compared with extracts depleted of cyclin A alone. Depletion of cyclin E therefore does not have a significant inhibitory effect on DNA replication. To demonstrate that the inhibition of DNA replication is indeed due to depletion of cyclin A and not due to depletion of another protein associated with cyclin A, we tested the ability of CDK2-associated cyclin A kinase to restore DNA replication in cyclin A-depleted cell extracts. Fig. 3 shows that purified cyclin A-CDK2 kinase fully rescues DNA replication in depleted extracts and brings replication to the level observed in control cell extracts. In contrast, no more than 1-2 pmol of dCMP are incorporated in a control experiment, where addition of cyclin A is performed in the absence of T antigen. This low level of incorporation probably represents repair. DNA replication was reconstituted by the addition of exogenous cyclin A-CDK2 kinase (final specific activity 98.2 pmol/20 min/18 μl) to cyclin A-depleted extracts. The kinase activity of cyclin A-depleted extracts is 10.3 pmol/20 min/18 μl. The addition of kinase activity that fully restores replication is less than the activity measured in control extracts (240 pmol/20 min/18 μl). These results indicate that replication can be fully reconstituted by kinase activity that is substantially below control levels. We also tested whether purified GST-cyclin A alone could rescue DNA replication activity of cyclin A-depleted cell extracts. Fig. 4A shows that the inhibition of DNA replication was fully rescued by addition of cyclin A at a concentration of 80 nM. Furthermore, the addition of cyclin A to cell extracts depleted of cyclin A restores cyclin A associated H-1 kinase activity (Fig. 4B). DNA replication in cyclin A-depleted extracts is fully restored by kina" @default.
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• W2020494268 date "1996-12-01" @default.
• W2020494268 modified "2023-09-30" @default.
• W2020494268 title "Role for Cyclin A-dependent Kinase in DNA Replication in Human S Phase Cell Extracts" @default.
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