Proliferating Human Cells Hypomorphic for Origin Recognition Complex 2 and Pre-replicative Complex Formation Have a Defect in p53 Activation and Cdk2 Kinase Activation
2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m507150200
ISSN1083-351X
AutoresJamie K. Teer, Yuichi Machida, Hélène Labit, Olivia Novac, Olivier Hyrien, Kathrin Marheineke, Maria Zannis‐Hadjopoulos, Anindya Dutta,
Tópico(s)RNA modifications and cancer
ResumoThe Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Δ/–) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2. The Origin Recognition Complex (ORC) is a critical component of replication initiation. We have previously reported generation of an Orc2 hypomorph cell line (Δ/–) that expresses very low levels of Orc2 but is viable. We have shown here that Chk2 is phosphorylated, suggesting that DNA damage checkpoint pathways are activated. p53 was inactivated during the derivation of the Orc2 hypomorphic cell lines, accounting for their survival despite active Chk2. These cells also show a defect in the G1 to S-phase transition. Cdk2 kinase activation in G1 is decreased due to decreased Cyclin E levels, preventing progression into S-phase. Molecular combing of bromodeoxyuridine-labeled DNA revealed that once the Orc2 hypomorphic cells enter S-phase, fork density and fork progression are approximately comparable with wild type cells. Therefore, the low level of Orc2 hinders normal cell cycle progression by delaying the activation of G1 cyclin-dependent kinases. The results suggest that hypomorphic mutations in initiation factor genes may be particularly deleterious in cancers with mutant p53 or increased activity of Cyclin E/Cdk2. DNA replication requires the action of numerous protein complexes to accurately and completely duplicate the genome in a timely fashion. In most organisms, a series of steps must occur to load the replication machinery onto the chromatin. This sequence of events has been examined in several model systems and is fairly well conserved (for review, see Ref. 1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar). Briefly, the Origin Recognition Complex (ORC), 2The abbreviations used are: ORC, origin recognition complex; RC, replicative complex; FACS, fluorescence-activated cell sorting; BrdUrd, bromodeoxyuridine; Pipes, 1,4-piperazinediethanesulfonic acid; CDK, cyclin-dependent kinase.2The abbreviations used are: ORC, origin recognition complex; RC, replicative complex; FACS, fluorescence-activated cell sorting; BrdUrd, bromodeoxyuridine; Pipes, 1,4-piperazinediethanesulfonic acid; CDK, cyclin-dependent kinase. a complex of six subunits, is thought to bind DNA, thus marking a replication origin (2Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (992) Google Scholar). ORC recruits Cdc6 and Cdt1 to the chromatin, both of which are required for loading the Mcm2–7 complex (3Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 4Cocker J.H. Piatti S. Santocanale C. Nasmyth K. Diffley J.F. Nature. 1996; 379: 180-182Crossref PubMed Scopus (293) Google Scholar, 5Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 6Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (292) Google Scholar, 7Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (367) Google Scholar). The Mcm2–7 complex is thought to be the helicase responsible for unwinding the double-stranded DNA (8Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 9Lee J.K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 10Chong J.P. Hayashi M.K. Simon M.N. Xu R.M. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1530-1535Crossref PubMed Scopus (259) Google Scholar, 11Schwacha A. Bell S.P. Mol. Cell. 2001; 8: 1093-1104Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12Blow J.J. Dutta A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 476-486Crossref PubMed Scopus (526) Google Scholar), allowing polymerases access to the DNA. The loading of Mcm2–7 completes formation of the pre-Replicative Complex (pre-RC), which must occur before the end of G1. At this stage, origins are "primed" and are awaiting the activity of cyclin-dependent kinases and Dbf4/Cdc7 kinase. These kinase activities are required to load downstream factors that make up the Pre-initiation Complex, Cdc45/Sld3, Mcm10, Dpb11/Sld2, and GINS. These proteins are thought to be important for polymerase loading by mechanisms that are not fully understood (reviewed in Refs. 1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar and 13Takeda D.Y. Dutta A. Oncogene. 2005; 24: 2827-2843Crossref PubMed Scopus (155) Google Scholar). The ORC itself is of great interest, as it defines the region in the genome where replication will begin. This function is vital, as evenly spaced initiations are important to complete replication of the entire genome as quickly as possible. In fact, ORC was initially identified in budding yeast based on its ability to interact with the yeast origin sequence (ARS) (2Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (992) Google Scholar). ORC is formed of six subunits, Orc1– 6, that range in size from 97 to 28 kDa. It has been shown that the ORC subunits interact with each other, albeit with different affinities (14Dhar S.K. Delmolino L. Dutta A. J. Biol. Chem. 2001; 276: 29067-29071Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 15Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). These studies show that Orc2 and Orc3 seem to form a core subcomplex with which other ORC members interact. Early studies on the ORC proteins showed that Orc1 (16Li J.J. Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (364) Google Scholar), Orc2 (17Micklem G. Rowley A. Harwood J. Nasmyth K. Diffley J.F. Nature. 1993; 366: 87-89Crossref PubMed Scopus (196) Google Scholar, 18Foss M. McNally F.J. Laurenson P. Rine J. Science. 1993; 262: 1838-1844Crossref PubMed Scopus (264) Google Scholar), Orc3 (19Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. Arkin A.P. Astromoff A. El-Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Guldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kotter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Nature. 2002; 418: 387-391Crossref PubMed Scopus (3222) Google Scholar), Orc4 (19Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. Arkin A.P. Astromoff A. El-Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Guldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kotter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Nature. 2002; 418: 387-391Crossref PubMed Scopus (3222) Google Scholar), Orc5 (19Giaever G. Chu A.M. Ni L. Connelly C. Riles L. Veronneau S. Dow S. Lucau-Danila A. Anderson K. Andre B. Arkin A.P. Astromoff A. El-Bakkoury M. Bangham R. Benito R. Brachat S. Campanaro S. Curtiss M. Davis K. Deutschbauer A. Entian K.D. Flaherty P. Foury F. Garfinkel D.J. Gerstein M. Gotte D. Guldener U. Hegemann J.H. Hempel S. Herman Z. Jaramillo D.F. Kelly D.E. Kelly S.L. Kotter P. LaBonte D. Lamb D.C. Lan N. Liang H. Liao H. Liu L. Luo C. Lussier M. Mao R. Menard P. Ooi S.L. Revuelta J.L. Roberts C.J. Rose M. Ross-Macdonald P. Scherens B. Schimmack G. Shafer B. Shoemaker D.D. Sookhai-Mahadeo S. Storms R.K. Strathern J.N. Valle G. Voet M. Volckaert G. Wang C.Y. Ward T.R. Wilhelmy J. Winzeler E.A. Yang Y. Yen G. Youngman E. Yu K. Bussey H. Boeke J.D. Snyder M. Philippsen P. Davis R.W. Johnston M. Nature. 2002; 418: 387-391Crossref PubMed Scopus (3222) Google Scholar), and Orc6 (16Li J.J. Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (364) Google Scholar) are all essential in yeasts. Although ORC is expected to be essential for human chromosomal replication, a detailed study of chromosomal replication with low levels of human ORC has not yet been published. Recent reports using small interfering RNA indicate that acute depletion of an ORC subunit leads to cell cycle arrest, making it impossible to study chromosomal replication under such conditions (20Prasanth S.G. Prasanth K.V. Siddiqui K. Spector D.L. Stillman B. EMBO J. 2004; 23: 2651-2663Crossref PubMed Scopus (206) Google Scholar, 21Machida Y.J. Teer J.K. Dutta A. J. Biol. Chem. 2005; 280: 27624-27630Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To study ORC in human cells, we have previously generated an Orc2 hypomorph cell line, in which Orc2 is only being expressed from one allele at 10% of wild type levels (22Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). This Orc2 Δ/– cell line (containing one hypomorph Orc2 allele and one null Orc2 allele) was found to be viable, presumably because of compensatory changes in other genes, and was able to proliferate for many generations. The low levels of Orc2 did, however, prevent replication of an exogenous plasmid containing a single Epstein-Barr virus origin. We believe that this difference in replication ability stems from a threshold effect; the levels of Orc2 were high enough to support chromosome replication, but not episome replication. In this study, we have examined the effects of low Orc2 on the replication of the cellular chromosomes, as well as the cell as a whole. Cell Culture—HCT116, HCT116 p53–/–, and Δ/– (HCT116 Orc2 Δ/–) cells were maintained in McCoy's 5A medium (Cellgro), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Standard tissue culture growth conditions and methods were used. Western, Northern, and RT-PCR—Western blots were performed as described. In this case, cells were lysed in 50 mm Tris, pH 7.4, 0.2% Nonidet P-40, 150 or 300 mm NaCl, 1 mm EDTA, 10 mm NaF, 0.2 mm Na3VO3, 1 mm phenylmethylsulfonyl fluoride, 2 mm dithiothreitol, and 1/100 Protease Inhibitor Mixture (Sigma). Orc3, -4, and -6, Cdt1, and RPA34 and RPA70 antibodies have been previously described (23Pinto S. Quintana D.G. Smith P. Mihalek R.M. Hou Z.H. Boynton S. Jones C.J. Hendricks M. Velinzon K. Wohlschlegel J.A. Austin R.J. Lane W.S. Tully T. Dutta A. Neuron. 1999; 23: 45-54Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 24Quintana D.G. Hou Z. 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, 25Dhar S.K. Dutta A. J. Biol. Chem. 2000; 275: 34983-34988Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 26Wohlschlegel J.A. Dwyer B.T. Dhar S.K. Cvetic C. Walter J.C. Dutta A. Science. 2000; 290: 2309-2312Crossref PubMed Scopus (577) Google Scholar). Rabbit antibodies against Orc5 were raised using His6-tagged recombinant Orc5-(75–686). p27 (C-19), p21 (C-19), and MCM7 antibodies were purchased from Santa Cruz Biotechnology, and Orc2 antibody was purchased from BD Biosciences. p53 antibody (D0–1) was purchased from Oncogene Science. Cdk2, Cyclin E, Cdk4, and Cdc25A (F-6) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Rb, pSer780-Rb, pSer807/811-Rb, pThr68-Chk2, and p-Ser216 Cdc25C were from Cell Signaling. pTyr15 Cdk2 was detected by immunoprecipitation against Cdk2, followed by Western using pTyr15 Cdc2 from Cell Signaling (recognizes pTyr15 on Cdc2 and Cdk2.) Chk2 was purchased from Sigma, and E2F1 was purchased from Upstate. Total cellular RNA was prepared using the RNAeasy Midi or Mini kit (Qiagen) or TRIzol extraction. Pulse-Chase—Logarithmic cells were starved of methionine for 1 h in Dulbecco's modified Eagle's medium, supplemented with dialyzed fetal bovine serum. Cells were then labeled with 300 μCi of [35S]methionine for 4 h. Cells were washed to remove unincorporated methionine and were chased with McCoy's 5A medium supplemented with fetal bovine serum for the indicated lengths of time. Cells were lysed in the same buffer as described above, but with 300 mm NaCl. Orc3 was immunoprecipitated with the above antibody and separated by SDS-PAGE. The gel was then dried and exposed to a phosphorimaging plate for visualization and analysis. FACS Analysis—Cells were prepared as previously described (27Vaziri C. Saxena S. Jeon Y. Lee C. Murata K. Machida Y. Wagle N. Hwang D.S. Dutta A. Mol. Cell. 2003; 11: 997-1008Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). The analysis was carried out on a BD Biosciences FACS Calibur using Cellquest and FloJo software. Chromatin Immunoprecipitation—In vivo cross-linking was performed as described in Ref. 28Ritzi M. Baack M. Musahl C. Romanowski P. Laskey R.A. Knippers R. J. Biol. Chem. 1998; 273: 24543-24549Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar with some modifications. In brief, 80% confluent HeLa, HCT116, or HCT116 Δ/– cells were grown as described above and then treated with formaldehyde (1%). Cross-linked cell nuclei were sonicated 10 times for 30 s each time, and the chromatin size was monitored by electrophoresis (29Hecht A. Grunstein M. Methods Enzymol. 1999; 304: 399-414Crossref PubMed Scopus (151) Google Scholar). This treatment generated fragments of ∼20 kb. To further reduce the chromatin size to smaller fragments of 1.5–3.5 kb, DNA was digested with SphI, HindIII, PstI, and EcoRI restriction endonucleases in NEB2 buffer (100 units of each; New England Biolabs, Beverly, MA) at 37 °C for 6 h. Sheared chromatin-lysed extracts were incubated with 50 μl of protein G-agarose (Roche Applied Science) to reduce background caused by nonspecific adsorption of irrelevant cellular proteins/DNA to proteins. These cleared chromatin lysates were incubated at 4 °C for 6 h on a rocker platform with either 50 μl of preimmune rabbit serum (Santa Cruz Biotechnology) or 5 μg of anti-Orc2, anti-Orc3, anti-Orc4, or anti-Orc6 antibodies. Protein G-agarose (50 μl) was added, and the incubation was continued for 12 h. The precipitates were successively washed two times for 5 min with 1 ml of each buffer, lysis buffer, WB1 (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.1% Nonidet P-40, 0.05% sodium deoxycholate), WB2 (as WB1 with no NaCl), and 1 ml of TE (20 mm Tris-HCl, pH 8.0, 1 mm EDTA). The precipitates were finally resuspended in 200 μl of extraction buffer (1% SDS/TE). The samples were incubated at 65 °C overnight to reverse the protein/DNA cross-links, followed by a 2-h incubation at 37 °C with 100 μg of proteinase K (Roche Applied Science). Finally, the samples were processed for DNA purification by passing them through QIAquick PCR purification columns (Qiagen, Valencia, CA). PCR reactions were carried out in 20 μl with 1/200th of the immunoprecipitated material with the use of LightCycler capillaries (Roche Applied Science) and the LightCycler-FastStart DNA Master SYBR Green I (Roche Applied Science). The real-time PCR quantification was performed as described in Ref. 30Ladenburger E.M. Keller C. Knippers R. Mol. Cell Biol. 2002; 22: 1036-1048Crossref PubMed Scopus (130) Google Scholar, using primer sets LB2 and LB2 C1. Cell Cycle Analysis—Cells were arrested in 40 ng/ml of nocodazole for 16 h. To release the cells from mitosis, they were washed three times in sterile phosphate-buffered saline and replated in fresh warmed medium. After the desired incubation time, cells were labeled with 10 μm BrdUrd for 1 h and then prepared for FACS or Western blot analysis as above. Alternately, after the desired incubation time, cells were treated with 1.25 μCi of [3H]thymidine for 1 h. Cells were then washed and incubated with cold stop solution (10% trichloroacetic acid, 200 mm sodium pyrophosphate). Cells were washed with 95% ethanol and solubilized (1% SDS, 10 mm NaOH.) The resulting solution was spotted on Whatman paper, dried, and counted on a Beckman LS 6000 scintillation counter. To examine cells in S-phase, cells were treated with 2 mm thymidine for 12 h, released into untreated medium for 12 h, and further treated with 1 μg/ml of aphidicolin for 12 h. Chromatin Fractionation—Chromatin fractions were isolated as described previously (31Todorov I.T. Attaran A. Kearsey S.E. J. Cell Biol. 1995; 129: 1433-1445Crossref PubMed Scopus (204) Google Scholar). Briefly, cells (2 × 106) were lysed in 100 μl of CSK buffer (10 mm Pipes, pH 7.0, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2) containing 0.5% Triton X-100, 1 mm ATP, 1 mm Na3VO4. Lysates were incubated on ice for 20 min and then centrifuged at 1500 rpm for 5 min at 4 °C. Supernatant (S1) was removed, and pellets were washed with 1 ml of lysis buffer and centrifuged again. Pellets were incubated in 100 μl of lysis buffer containing 1 mm CaCl2 and 120 units of micrococcus nuclease (Worthington) for 10 min at 37 °C and centrifuged. Supernatant (S2, chromatin-bound fraction) was removed, and pellets were washed with 1 ml of lysis buffer and centrifuged again. Pellets were boiled in 100 μl of 1× sample buffer (P2). DNA Combing and Detection by Fluorescent Antibodies—Cells were synchronized at very early S-phase by treatment with 2 mm thymidine for 12 h, release into untreated medium for 12 h, and further treatment with 1 μg/ml of aphidicolin for 12 h. Cells were released into medium containing 50 μm BrdUrd for the indicated time points. Cells were then washed in medium containing 100 μm thymidine and incubated in 10 μm thymidine until 8 h post release. Cells were then harvested with trypsin/EDTA and suspended in 1% agarose. Agarose blocks were incubated with ESPK (0.5 m EDTA, 1% sarkosyl, 2 mg/ml proteinase K (Fisher)) for 24 h twice. Blocks were then washed with TE/phenylmethylsulfonyl fluoride and stored in 0.5 m EDTA. DNA from whole cells was extracted and combed on silanised coverslips as described (32Michalet X. Ekong R. Fougerousse F. Rousseaux S. Schurra C. Hornigold N. van Slegtenhorst M. Wolfe J. Povey S. Beckmann J.S. Bensimon A. Science. 1997; 277: 1518-1523Crossref PubMed Scopus (513) Google Scholar). Combed DNA was dehydrated in a series of ethanol (70, 90, and 100%), denatured with 1N NaOH for 30 min, again dehydrated, and blocked in a blocking solution (1× phosphate-buffered saline, 0.1% Tween, 1% bovine serum albumin) for 1 h. BrdUrd was detected with an anti-BrdUrd antibody (Abcys), followed by an anti-rat antibody conjugated with AlexaFluor 488 and an anti-goat AlexaFluor 488 antibody (33Marheineke K. Hyrien O. J. Biol. Chem. 2004; 279: 28071-28081Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Total DNA was visualized afterward by an anti-guanosine antibody (Argene), followed by an anti-mouse AlexaFluor 594 (Molecular Probes). Antibody incubations were in general for 30 min and were separated by three-four washes with 1× phosphate-buffered saline, 0.1% Tween. Coverslips were mounted in Vectashield® solution. To analyze data, images of the combed DNA molecules were acquired by a Leitz DC300F camera associated with the LeicaFW4000 software and measured by ImageGauge 4.2 software. Fields of view were chosen at random in the AlexaFluor 594 channel and then photographed under the AlexaFluor 594 and AlexaFluor 488 filters. The replication extent of each sample was defined as the sum of all eye lengths divided by the total length of the molecules. Fork density is the total number of forks divided by total DNA length (kb) in each sample, as detailed in Ref. 33Marheineke K. Hyrien O. J. Biol. Chem. 2004; 279: 28071-28081Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar. Total DNA length in each case was normalized using the number of cells entering S-phase as shown in Fig. 5D). Absence of Orc2 Affects Core ORC Complex Stability—A hypomorph mutation of Orc2 in HCT116 cells (which we designate the Δ/– cell line) decreases Orc2 protein level by 90% (22Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). We also observed decreased levels of Orc3 and Orc5 protein even though their genomic loci remained unaltered (Ref. 22Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar and data not shown.) Orc1, Orc4, Orc6, and Cdt1 protein levels are unaffected in Δ/–, indicating that the Orc2,3,5 subcomplex alone is affected by the lack of Orc2. mRNA levels of Orc3 and Orc5 were unchanged in the Δ/– cells (supplemental Fig. S1), suggesting that the protein decrease is due to another mechanism after transcription. Orc3 and Orc5 proteins might be destabilized due to low levels of the Orc3 binding partner. To examine this, Orc3 stability was determined by a [35S]methionine-labeling pulse-chase experiment. Orc3 was less stable in the Δ/– cells (Fig. 1A), and quantification of the fluorogram revealed a marked decrease in half-life: 11 h in HCT116 cells, compared with 6 h in the Δ/– cells (Fig. 1B). It is possible that the destabilization results from proteasomal degradation. However, Orc3 was not stabilized in either cell line after MG132 treatment, in contrast to p27, a known target of the proteasome (34Pagano M. Tam S.W. Theodoras A.M. Beer-Romero P. Del Sal G. Chau V. Yew P.R. Draetta G.F. Rolfe M. Science. 1995; 269: 682-685Crossref PubMed Scopus (1734) Google Scholar) (Fig. 1C). Therefore, Orc3 is not targeted for proteasomal degradation. Similar results were observed for Orc5 (data not shown). These results suggest that the decrease in Orc3 protein level is due to destabilization in the absence of the partner protein Orc2 by mechanisms that do not depend on the proteasome. This destabilization of the Orc2,3,5 subcomplex may adversely affect pre-RC loading in the Δ/– cells. ORC and Pre-RC Loading Is Decreased in Orc2 Hypomorph Cells—In yeast, Orc2 is required for DNA replication initiation (35Bell S.P. Genes Dev. 2002; 16: 659-672Crossref PubMed Scopus (238) Google Scholar). To investigate ORC loading at a specific origin, chromatin immunoprecipitation was performed using several ORC subunits. The associated chromatin was assayed by quantitative PCR using primers in the Lamin B2 region, a previously reported human origin (36Giacca M. Zentilin L. Norio P. Diviacco S. Dimitrova D. Contreas G. Biamonti G. Perini G. Weighardt F. Riva S. Falaschi A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7119-7123Crossref PubMed Scopus (174) Google Scholar). Orc2, 3, 4, and 6 associated with the Lamin B2 origin in HeLa cells, but not a nearby control region, whereas normal rabbit serum did not associate with either region (Fig. 2A). The experiment was then performed on HCT116 +/+ and Δ/– cells, which revealed a decrease of ORC loading on Lamin B2 in Δ/– cells (Fig. 2B). The amount of cross-linked DNA molecules pulled down in HCT116 cells was 3- to 7.7-fold higher than in the Δ/– cells. This indicates that origin association of the entire ORC (including Orc4 and Orc6, whose levels are not changed) is dependent on the presence of Orc2. This extends our earlier observation that Orc4 loading decreases in a chromatin fractionation experiment (22Dhar S.K. Yoshida K. Machida Y. Khaira P. Chaudhuri B. Wohlschlegel J.A. Leffak M. Yates J. Dutta A. Cell. 2001; 106: 287-296Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Interestingly, origin specificity (the ratio of ORC signal at Lamin B2 versus the control region) was retained in Δ/– cells despite the low levels of ORC binding. ORC association with origins recruits Cdt1 and Cdc6, which in turn recruit the MCM2–7 complex to complete a functional pre-RC (5Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 6Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (292) Google Scholar). To investigate pre-RC loading, we fractionated cells and examined MCM7 levels in the chromatin fractions. Chromatin-bound MCM7 was drastically decreased in the Δ/– cells (Fig. 2c), which indicates that MCM7 loading, and thus pre-RC formation, is dependent on ORC loading. (The use of p53–/– cells as a further control is explained later.) The decrease in pre-RC loading suggests that replication may be affected in the Δ/– cells, as chromatin-loaded MCM2–7 is required not only to recruit additional factors for replication but also to unwind the DNA for fork firing. Chk2 Is Phosphorylated in Δ/– Cells—The low levels of pre-RC are expected to lead to replication stress. This could cause fork stalling (and checkpoint activation) as forks extend from more sparsely firing origins. To test for checkpoint activation, we looked at phosphorylation of Chk1 at serine 317, a target of the DNA damage modulator ATR (37Zhao H. Piwnica-Worms H. Mol. Cell Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (852) Google Scholar). We also looked at phosphorylation of Chk2 at threonine 68, a target of the other major modulator, ATM (38Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (683) Google Scholar). Chk1 phosphorylation, although induced by hydroxyurea, is not increased in Δ/– cells (data not shown.) However, we found that Chk2 phosphorylation at Thr-68 is increased in the Δ/– cells (Fig. 3A). It therefore appears that a DNA damage checkpoint is activated in these cells. To determine the downstream effects of Chk2 phosphorylation, we examined several known targets. Cdc25C is phosphorylated by Chk2 at serine 216 (39Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1082) Google Scholar, 40Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (358) Google Scholar), which serves to inhibit its normal role in activating Cyclin B/Cdc2 (41Blasina A. de Weyer I.V. Laus M.C. Luyten W.H. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), thus preventing mitotic entry. Phosphorylation of Cdc25C was slightly decreased in Δ/– cells, indicating it is not a target of the active Chk2 (supplemental Fig. S2a). Cdc25A is also phosphorylated by Chk2, causing its degradation (42Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (868) Google Scholar), which, in turn, would lead to increased Tyr-15 phosphorylation on Cdk2 (43Hoffmann I. Draetta G. Karsenti E. EMBO J. 1994; 13: 4302-4310Crossref PubMed Scopus (422) Google Scholar, 44Blomberg I. Hoffmann I. Mol. Cell. Biol. 1999; 19: 6183-6194Crossref PubMed Scopus (253) Google Scholar). We did not, however, observe any decrease in the amount of Cdc25A in the Δ/– cells (it was actually increased) (supplemental Fig. S2a). Likewise, the total amount of Tyr-15 phosphorylation on Cdk2 was less in Δ/– cells, the opposite of what is expected if Cdc25A were inactive (supplemental Fig. S2b). Therefore, Cdc25A and the inhibitory phosphorylation of Cdk2 do not seem to be affected by activated Chk2 in the Δ/– cells. Perhaps the most well studied target of Chk2 is p53. Chk2 stabilizes p53 by phosphorylating it at serine 20 (45Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar, 46Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1044) Google Scholar). We were surprised to see that p53 was completely absent in these cells at both the protein and message level (Fig. 3, B and C). Induction with γ irradiation failed to
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