The ORC1 Cycle in Human Cells

Abstract

Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2–5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring. Components of ORC (the origin recognition complex) are highly conserved among eukaryotes and are thought to play an essential role in the initiation of DNA replication. The level of the largest subunit of human ORC (ORC1) during the cell cycle was studied in several human cell lines with a specific antibody. In all cell lines, ORC1 levels oscillate: ORC1 starts to accumulate in mid-G1 phase, reaches a peak at the G1/S boundary, and decreases to a basal level in S phase. In contrast, the levels of other ORC subunits (ORCs 2–5) remain constant throughout the cell cycle. The oscillation of ORC1, or the ORC1 cycle, also occurs in cells expressing ORC1 ectopically from a constitutive promoter. Furthermore, the 26 S proteasome inhibitor MG132 blocks the decrease in ORC1, suggesting that the ORC1 cycle is mainly due to 26 S proteasome-dependent degradation. Arrest of the cell cycle in early S phase by hydroxyurea, aphidicolin, or thymidine treatment is associated with basal levels of ORC1, indicating that ORC1 proteolysis starts in early S phase and is independent of S phase progression. These observations indicate that the ORC1 cycle in human cells is highly linked with cell cycle progression, allowing the initiation of replication to be coordinated with the cell cycle and preventing origins from refiring. The replication of chromosomal DNA in eukaryotes is limited to once per cell division cycle. This control appears to be achieved mainly by the regulation of replication origins so that they fire only once per cell cycle. The origin recognition complex (ORC), 1The abbreviations used are: ORC, origin recognition complex; MCM, mini-chromosome maintenance; pre-RC, pre-replicative complex; FCS, fetal calf serum; PBS, phosphate-buffered saline; HU, hydroxyurea; DAPI, 4,6-diamidino-2-phenylindole; CMV, cytomegalovirus; Pipes, 1,4-piperazinediethanesulfonic acid; TBS, Tris-buffered saline; LC/MS/MS, liquid chromatography-tandem mass spectrometry; FACS, fluorescence-activate cell sorting; BrdUrd, 5-bromo-2′-deoxy-uridine. identified in budding yeast as a protein complex that binds origins, consists of six gene products (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (1030) Google Scholar, 2Diffley J.F. Cocker J.H. Nature. 1992; 357: 169-172Crossref PubMed Scopus (304) Google Scholar, 3Li J.J. Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (370) Google Scholar, 4Diffley J.F. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 5Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (320) Google Scholar). In addition to ORC, several factors highly conserved among eukaryotes are involved in initiation (6Gavin K.A. Hidaka M. Stillman B. Science. 1995; 270: 1667-1671Crossref PubMed Scopus (206) Google Scholar, 7Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1410) Google Scholar, 8Depamphilis M.L. Gene (Amst.). 2003; 22: 1-15Crossref Scopus (118) Google Scholar). The sequential assembly of these factors on origin-ORC complexes precedes initiation, as has been shown in yeast and Xenopus systems. For example, mini-chromosome maintenance (MCM) proteins are loaded onto origins in the presence of ORC and CDC6, which establishes the pre-replicative complex (pre-RC) necessary for subsequent protein assembly (9Carpenter P.B. Mueller P.R. Dunphy W.G. Nature. 1996; 379: 357-360Crossref PubMed Scopus (176) Google Scholar, 10Donovan S. Harwood J. Drury L.S. Diffley J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (435) Google Scholar, 11Fujita M. Hori Y. Shirahige K. Tsurimoto T. Yoshikawa H. Obuse C. Genes Cells. 1998; 3: 737-749Crossref PubMed Scopus (22) Google Scholar, 12Tadokoro R. Fujita M. Miura H. Shirahige K. Yoshikawa H. Tsurimoto T. Obuse C. J. Biol. Chem. 2002; 277: 15881-15889Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 13Takahashi T. Ohara E. Nishitani H. Masukata H. EMBO J. 2003; 22: 964-974Crossref PubMed Scopus (50) Google Scholar). After origin firing, the pre-RC changes to a post-replicative form by the dissociation of MCM from the complex (12Tadokoro R. Fujita M. Miura H. Shirahige K. Yoshikawa H. Tsurimoto T. Obuse C. J. Biol. Chem. 2002; 277: 15881-15889Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 14Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 15Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). These associations provide an important mechanism that 1) ensures that replication origins fire at precise times and 2) prevents re-initiation. Budding yeast ORC is a static complex that is maintained at a constant level and remains bound to origins throughout the cell cycle (4Diffley J.F. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 5Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (320) Google Scholar). Thus, MCM loading in yeast is mainly regulated by other factors such as cell cycle-regulated Cdc6 or CDK kinase activities (7Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1410) Google Scholar, 8Depamphilis M.L. Gene (Amst.). 2003; 22: 1-15Crossref Scopus (118) Google Scholar, 16Nguyen V.Q. Co C. Li J.J. Nature. 2001; 411: 1068-1073Crossref PubMed Scopus (363) Google Scholar). It is also known that the phosphorylation status of ORC subunits correlates with the timing of pre-RC formation, suggesting a role for ORC phosphorylation in MCM loading (16Nguyen V.Q. Co C. Li J.J. Nature. 2001; 411: 1068-1073Crossref PubMed Scopus (363) Google Scholar). Similar phosphorylation of ORC subunits was found in a Xenopus egg extract system, suggesting a conserved mechanism for the regulation of ORC functions (17Romanowski P. Marr J. Madine M.A. Rowles A. Blow J.J. Gautier J. Laskey R.A. J. Biol. Chem. 2000; 275: 4239-4243Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Tugal T. Zou-Yang X.H. Gavin K. Pappin D. Canas B. Kobayashi R. Hunt T. Stillman B. J. Biol. Chem. 1998; 273: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Recent studies have elucidated possible mechanisms for the regulation of ORC activity in mammals. In human and hamster cells, ORC1, the largest subunit, is ubiquitinated in S phase (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 20Fujita M. Ishimi Y. Nakamura H. Kiyono T. Tsurumi T. J. Biol. Chem. 2002; 277: 10354-10361Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar). In human cells, ORC1 is poly-ubiquitinated by the SCFskp2 ubiquitin ligase complex and degraded through the 26 S proteasome pathway (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Indeed, the cellular content of ORC1 is greatly reduced in S phase (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 22Kreitz S. Ritzi M. Baack M. Knippers R. J. Biol. Chem. 2001; 276: 6337-6342Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In hamster cells, ORC1 is only mono-ubiquitinated and may be regulated by a mechanism other than degradation (21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar). Indeed, the cellular ORC1 level remains constant in hamster cells (21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar, 23Okuno Y. McNairn A.J. den Elzen N. Pines J. Gilbert D.M. EMBO J. 2001; 20: 4263-4277Crossref PubMed Scopus (113) Google Scholar). These data suggest that the activity of mammalian ORC is regulated by ubiquitination of ORC1, but ubiquitinated ORC1 has different fates in different species or cell lines. To elucidate the mechanisms that regulate ORC activity in mammals, and especially in humans, we determined the levels of ORC subunits in several human cell lines throughout the cell cycle. From systematic studies in which we synchronized these cells by a variety of methods, we conclude that ORC1 exhibits cell cycle-dependent oscillation, a phenomenon we have termed the ORC1 cycle. The ORC1 cycle ensures the dynamic assembly of ORC and MCM on chromatin in G1 phase, which may correspond to the formation of the pre-RC in human cells, as demonstrated in an accompanying paper (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Cell Culture and Synchronization—Cells were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum (FCS). To impose cell cycle arrest by the double-thymidine block method, HeLa S3 cells were incubated in the presence of 2.5 mm thymidine for two periods of 24 h with an intervening incubation of 12 h in the absence of thymidine. For M phase arrest, cells were subjected to the double-thymidine block procedure and then cultured in the presence of 150 ng/ml TN16 (WAKO, Japan) for 12 h. Synchronous growth following arrest was achieved by two washes with PBS and subsequent culturing without reagents. Telomerase-immortalized human retina pigment epithelial cells (TERT-RPE1 cells, Invitrogen) were synchronized by serum starvation. TERT-PRE1 cells (50% confluent) were incubated with Dulbecco's modified Eagle's medium without FCS for 84–96 h and then released from starvation by the addition of 10% FCS. Hydroxyurea (HU) was included at 2.5 mm. Synchronized growth was monitored by flow of cells Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google by of and and by was the and cells were with for and a and a as by the were also with as Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The were with a and with of in ORC1 with a to the and an to an was of the of The was cells with and expressing were by on expressing a level of to the level of ORC1 was for the shown in of Cell cell cells on were times with PBS, in modified mm mm mm mm mm mm and with an of mm mm and was as Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). from HeLa S3 cells were with as Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). human ORC1 and human were from with ORC1 protein with or with at The were and from by To the were with and and were from and and were by A. Dutta A. Dutta A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. Dutta A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). which with human has been H. N. K. EMBO J. 1994; PubMed Scopus Google Scholar). was from with these was as Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). with and the with the cells on were with PBS with in PBS for and times with The were incubated with Tris-buffered for times with incubated with in 10% FCS at times with and incubated with a with at for h. After washes with by PBS cells were with in PBS for and times with were with the in the were a to a or by an To and cells levels were the and cells were as observations of ORC1 and in HeLa S3 of HeLa S3 cells by phase or by with the (ORC1) and and ORC1 is and is are Cell cycle were determined by of for early and S are of ORC1 from Cell with ORC1 in human cells the which was a human ORC1 Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In we a which a as shown by and proteins of the from human cell (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). have that the that with is ORC1 by two lines of the of from the of human ORC1, as shown by (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). the shown in cells were subjected to with human (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). the is highly specific to human the other also with in human cells, ORC1, that also with the The recognition of may human ORC1 Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, we human ORC1 with the and that of are that we the ORC1 content of HeLa S3 cells to be and the ORC1 level in a cell cycle-dependent as we used the to ORC1 in human Cell of ORC1 in HeLa S3 Synchronized by M and for the we specific for that be used to in HeLa S3 cell HeLa S3 cells released from a arrest by treatment with Cell cycle was monitored by cells and by the These that S phase and h from arrest were from cells at and subjected to with ORC and E. The two proteins were used as for cell cycle E. Science. 1992; PubMed Scopus Google Scholar). It is that the level with the of cells and that the level in S phase. ORC1 was at a basal level from to early G1 to and at h at a level than the basal and then from S to phase In contrast, the levels of the subunits constant The oscillation of ORC1 by was by The of cells during G1 to the G1/S and during S phase These two indicate that the in ORC1 than the in the level and in the of cells, which at h The ORC1 S in precise observations of cells with the by that ORC1 as in HeLa S3 suggesting that with The of in a cell was with in the cell cycle by for in than were throughout the in G1 phase cells, but in with cell cycle early S and These data suggest that the decrease in ORC1 that precedes S phase is a of changes in protein levels in ORC1 with suggesting that ORC1 is at of DNA Cell of ORC1 we the oscillation of ORC1 in HeLa S3 cells is cell two human cell lines, cell with and cell demonstrated that cells S phase with a to that of HeLa S3 cells, but cells S phase as by and the of For cell lines, were and the ORC1 level than the in the of cells, at constant levels throughout the cell cycle, as for HeLa S3 cells and we oscillation be for ORC1 ectopically from a in For a cell expressing ORC1 at a level to that of ORC1 we oscillation in the levels of the protein as that of ORC1 a different to ORC1 HeLa and cells were in early S phase for 24 h by the double-thymidine block method, and cellular protein were studied as from arrest In all ORC1 was at a basal level at and the of was in M phase the ORC1 peak than the in the of the levels of were used a in the by cells in and with Thus, is possible that the decrease in ORC1 be due to degradation during ORC1 has been to be (21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar, 23Okuno Y. McNairn A.J. den Elzen N. Pines J. Gilbert D.M. EMBO J. 2001; 20: 4263-4277Crossref PubMed Scopus (113) Google Scholar). To we 1) cells in were with and 2) cells were with and then with In the of degradation of ORC1 in from cells was the as in by method, indicating that the levels of ORC1 in cell the levels of ORC1 in human studies with cells human retina pigment epithelial in by serum starvation for that the level of ORC1 was also in these cells the cells were with the level of ORC1 to at 12 at and in the following S phase This that the ORC1 cycle also occurs in synchronized of cell for and methods, the ORC1 cycle be in human Cell the ORC1 has been shown that are of the ORC1 gene and that ORC1 is cell cycle-regulated K. J. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar). This that of ORC1 may control ORC1 during G1 phase. we demonstrated that ORC1 from a indicating that control only a to the regulation of ORC1 levels in cells also that the reduced level of ORC1 in S cells was by treatment with a MG132 This that the ORC1 cycle the degradation of ORC1 by the S proteasome has been that ORC1 is poly-ubiquitinated by during S phase and degraded the 26 S proteasome pathway in human cells (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). ORC1 to to or in S ORC1 is at a basal level in cells in early S phase by the ORC1 level is in early S phase cells released from M phase The S phase degradation of ORC1 was also during arrest by (HU) or treatment These suggest that the degradation of ORC1 is the early S phase arrest the protein to decrease to a basal level in To the in the level of ORC1 in cells the early S phase arrest HeLa S3 cells were in M phase and then released in the presence of that these cells the from M to G1 and but a DNA content at indicating that they were in early S phase by these the ORC1 cycle was indicating that the of ORC1 degradation on to or at early S phase. the other an in the level of in the chromatin the in ORC1, bound to chromatin at levels ORC1 was degraded This is loaded onto chromatin in an (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google subsequent is independent of ORC1 degradation. It is known that the replication initiation ORC, remains at a constant level in chromatin throughout the cell cycle of species (4Diffley J.F. Cocker J.H. Dowell S.J. Rowley A. Cell. 1994; 78: 303-316Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 5Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (320) Google Scholar, 7Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1410) Google Scholar, 8Depamphilis M.L. Gene (Amst.). 2003; 22: 1-15Crossref Scopus (118) Google Scholar, P. J. Cell Sci. Google Scholar, D. DePamphilis M.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, DePamphilis M.L. EMBO J. 2002; PubMed Scopus Google Scholar). we have shown in different cell lines subjected to methods, human ORC1 exhibits a cell oscillation in as by and the other the other ORC remain at constant levels throughout the cell cycle. These observations have been for HeLa and cells by (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). these we conclude in human cells, the levels of ORC1 a phenomenon we have termed ORC1 M.L. Gene (Amst.). 2003; 22: 1-15Crossref Scopus (118) Google Scholar). studies that the level of ORC1 was constant throughout the human cell cycle Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. S.J. M. Dutta A. Mol. Cell. Biol. 1998; PubMed Scopus Google an that may be by the of the human ORC1 with other as we have demonstrated important the of of the ORC1 cycle during in yeast or have been to the of ORC1 in other is highly specific to human a ORC1 cycle has been in hamster in these cells, the level of ORC1 is constant (21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar, 23Okuno Y. McNairn A.J. den Elzen N. Pines J. Gilbert D.M. EMBO J. 2001; 20: 4263-4277Crossref PubMed Scopus (113) Google but the protein is released from the chromatin in S phase (21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar, DePamphilis M.L. EMBO J. 2002; PubMed Scopus Google Scholar). the level of ORC1 associated with chromatin in human and hamster cells, of cellular In with these the S ubiquitination of ORC1 has been for species (19Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 20Fujita M. Ishimi Y. Nakamura H. Kiyono T. Tsurumi T. J. Biol. Chem. 2002; 277: 10354-10361Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Li C.J. DePamphilis M.L. Mol. Cell. Biol. 2002; 22: 105-116Crossref PubMed Scopus (111) Google Scholar). hamster ORC1 is only but human ORC1 is and these may have for the different fates of the two ORC1 proteins in S phase. In cells, ORC1 is at a basal as for M or early S This basal level may be maintained by but regulation also have a role in the level of ORC1 in Indeed, of ORC1 in cells the of during M. EMBO J. PubMed Scopus Google Scholar). In of the ORC1 gene is at a basal level in cells, and be by the addition of serum or by of the K. J. Mol. Cell. Biol. 1996; PubMed Scopus Google suggesting the of regulation in the maintenance of a level of ORC1 during In ORC binds to replication origins as a complex of six proteins throughout the cell cycle, and in of these subunits in a in the initiation of suggesting that an ORC is necessary Y. Shirahige K. Obuse C. Tsurimoto T. Yoshikawa H. Mol. Cell. Biol. 1996; Scopus Google Scholar, S.P. Kobayashi R. Stillman B. Science. 1993; 262: PubMed Scopus Google Scholar). ORC is for human DNA the of ORC1 may be a in the regulation of ORC the levels of are appears to with only and are S. P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Dutta A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). we have the level of human ORC1 protein is cell cycle which in that ORC activity is by cell cycle progression. Indeed, we have demonstrated that formation of the complex the of ORC1 in human which is linked with the loading of MCM onto These are in the accompanying paper (24Ohta S. Tatsumi Y. Fujita M. Tsurimoto T. Obuse C. J. Biol. Chem. 2003; 278: 41535-41540Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). ORC1 is at a basal level in cells in early S phase by aphidicolin, HU, or thymidine. Furthermore, the cells DNA without ORC1 from Thus, we that ORC1 degradation to S phase. was for Xenopus ORC1 hamster were Xenopus egg DePamphilis M.L. EMBO J. 2002; PubMed Scopus Google Scholar). loaded onto hamster chromatin was released the assembly of on It has been also that is for the of S phase once have DePamphilis M.L. EMBO J. 2002; PubMed Scopus Google Scholar, X.H. J. J. Cell Biol. 1998; PubMed Scopus Google Scholar). Thus, is possible that a basal level of ORC1 is for origin we that ORC1 that has in G1 is used to origin and is necessary for subsequent that ORC1 and This the and the that the or degradation of ORC1 is necessary to the cell cycle to from the initiation to the It is important to the degradation of ORC1 to origin are the assembly of the pre-RC and the in or kinase studies of ORC at specific origins in human cells, be necessary to Masukata and Fujita for of the and are also to Dutta for