The ORC1 Cycle in Human Cells

The origin recognition complex (ORC) plays a central role in regulating the initiation of DNA replication in eukaryotes. The level of the ORC1 subunit oscillates throughout the cell cycle, defining an ORC1 cycle. ORC1 accumulates in G1 and is degraded in S phase, although other ORC subunits (ORCs 2–5) remain at almost constant levels. The behavior of ORC components in human cell nuclei with respect to the ORC1 cycle demonstrates that ORCs 2–5 form a complex that is present throughout the cell cycle and that associates with ORC1 when it accumulates in G1 nuclei. ORCs 2–5 are found in both nuclease-insoluble and -soluble fractions. The appearance of nuclease-insoluble ORCs 2–5 parallels the increase in the level of ORC1 associating with nuclease-insoluble, non-chromatin nuclear structures. Thus, ORCs 2–5 are temporally recruited to nuclease-insoluble structures by formation of the ORC1–5 complex. An artificial reduction in the level of ORC1 in human cells by RNA interference results in a shift of ORC2 to the nuclease-soluble fraction, and the association of MCM proteins with chromatin fractions is also blocked by this treatment. These results indicate that ORC1 regulates the status of the ORC complex in human nuclei by tethering ORCs 2–5 to nuclear structures. This dynamic shift is further required for the loading of MCM proteins onto chromatin. Thus, the pre-replication complex in human cells may be regulated by the temporal accumulation of ORC1 in G1 nuclei. The origin recognition complex (ORC) plays a central role in regulating the initiation of DNA replication in eukaryotes. The level of the ORC1 subunit oscillates throughout the cell cycle, defining an ORC1 cycle. ORC1 accumulates in G1 and is degraded in S phase, although other ORC subunits (ORCs 2–5) remain at almost constant levels. The behavior of ORC components in human cell nuclei with respect to the ORC1 cycle demonstrates that ORCs 2–5 form a complex that is present throughout the cell cycle and that associates with ORC1 when it accumulates in G1 nuclei. ORCs 2–5 are found in both nuclease-insoluble and -soluble fractions. The appearance of nuclease-insoluble ORCs 2–5 parallels the increase in the level of ORC1 associating with nuclease-insoluble, non-chromatin nuclear structures. Thus, ORCs 2–5 are temporally recruited to nuclease-insoluble structures by formation of the ORC1–5 complex. An artificial reduction in the level of ORC1 in human cells by RNA interference results in a shift of ORC2 to the nuclease-soluble fraction, and the association of MCM proteins with chromatin fractions is also blocked by this treatment. These results indicate that ORC1 regulates the status of the ORC complex in human nuclei by tethering ORCs 2–5 to nuclear structures. This dynamic shift is further required for the loading of MCM proteins onto chromatin. Thus, the pre-replication complex in human cells may be regulated by the temporal accumulation of ORC1 in G1 nuclei. The replication of eukaryotic chromosomes occurs in a highly regulated manner during S phase. In the budding yeast Saccharomyces cerevisiae the origin recognition complex (ORC), 1The abbreviations used are: ORC, origin recognition complex; pre-RC, pre-replicative complex; MCM, mini-chromosome maintenance; RNAi, RNA interference; siRNA, small interference RNA; LC/MS/MS, liquid chromatography-tandem mass spectrometry.1The abbreviations used are: ORC, origin recognition complex; pre-RC, pre-replicative complex; MCM, mini-chromosome maintenance; RNAi, RNA interference; siRNA, small interference RNA; LC/MS/MS, liquid chromatography-tandem mass spectrometry. which is composed of six polypeptides, is essential for initiation, and it remains bound to replication origins throughout the cell cycle (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (997) Google Scholar, 2Liang C. Stillman B. Genes Dev. 1997; 11: 3375-3386Crossref PubMed Scopus (318) Google Scholar, 3Diffley J.F. Cocker J.H. Dowell S.J. Rowley A. 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Despite the overall high conservation of ORC proteins among species, animal ORCs exhibit greater regulatory flexibility than their yeast counterparts. For example, expression of the mammalian ORC1 gene is regulated by the E2F protein (16Ohtani K. DeGregori J. Leone G. Herendeen D.R. Kelly T.J. Nevins J.R. Mol. Cell. Biol. 1996; 16: 6977-6984Crossref PubMed Scopus (128) Google Scholar, 30Asano M. Wharton R.P. EMBO J. 1999; 18: 2435-2448Crossref PubMed Scopus (87) Google Scholar), and Xenopus ORC is known to dissociate from metaphase chromatin, as observed in an egg extract system (11Romanowski P. Madine M.A. Rowles A. Blow J.J. Laskey R.A. Curr. Biol. 1996; 6: 1416-1425Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In addition, the Drosophila ORC1 protein changes in abundance depending on and on the cell cycle, and expression of the of This that ORC, and regulates the of animal cells by S phase M. Wharton R.P. EMBO J. 1999; 18: 2435-2448Crossref PubMed Scopus (87) Google Scholar). the Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) an ORC1 cycle in human cells, which is by ORC1 accumulation during G1 and reduction during the S phase. In this that subunits are recruited to nuclease-insoluble, non-chromatin structures in a manner that parallels the ORC1 cycle. this shift in the of ORCs 2–5 with the of MCMs to chromatin fractions. These results suggest that the accumulation of ORC1 replication complex in human nuclei to the S phase. and and of the cell cycle as Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). of the cell also Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of cell extract and of proteins from cells with as Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). RNA interference as by specific for ORC1 ORC1 CDC6 J. W. A. K. T. Nature. 2001; PubMed Scopus Google Scholar), and into cells with the and and used in this have been Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by of the to protein at a of of to of with ORC1 from extracts Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) from cells with of at for For the with of at for by of of protein and at for The as fractions. The with and with and with of as Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). as S. Y. K. Obuse Tsurimoto T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). by and the of the proteins from to at of in the to and with at for the with and and in from each by phase and to an mass with a on a The by the protein with the to high proteins. of ORC in the behavior of ORC subunits in human cells, proteins from cells into and fractions by with -3, and -5 found in the suggesting that they to chromatin. the nuclei further with to extract DNA Y. Tsurimoto T. Shirahige K. Yoshikawa H. Obuse C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), ORC1 with nuclease-insoluble nuclear structures nuclear In of the -3, and -5 subunits (ORCs 2–5) by this indicating that of are with nuclear structures the with of ORC1 by by ORCs 2–5 by and as shown in of the ORC in determine human ORC subunits are in a complex similar to yeast ORC, with ORC1 from the of cells with specific on of proteins with the the proteins in these from the to mass with ORC1 found in which proteins of with ORC2 and identified in and with and found in this to with specific ORCs 2–5 in which ORC1 in the of the proteins in the extract with This further by of ORC1 with an from the which the protein at the level as ORC1 Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ORCs 2–5 with These results indicate that a protein complex of the ORC1–5 subunits is present in the of human cell of the ORC1–5 as a complex throughout the cell cycle, as observed for yeast from cells from phase by the cellular ORC1–5 subunits with In this the level of ORC1 in metaphase and early G1 and a at and then from S to phase, in of the in the level of as in a Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) The of the however, constant The ORC1 at and at which it and it subunits in the and In a with the constant of both ORC2 and throughout the cell cycle and ORC1 in when it in cells. although the of and are constant throughout the cell cycle Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the of these proteins that with ORC2 when ORC1 present These results indicate that ORC2 and form a complex throughout the cell cycle and that their association with and is when ORC1 accumulates on chromatin and associates with the complex from G1 to early S phase. of ORCs 2–5 to a with ORC1 protein found in a non-chromatin nuclear in extracts from cells, ORCs 2–5 into both and fractions ORC1 to associate with ORCs 2–5 that ORCs 2–5 are recruited to the their association with determine the of ORCs 2–5 to the is cell and occurs in ORC1 and ORC2 from cells from by to G1 cellular ORC2 with I. ORC2 to appear in the at and at This temporal association of ORC2 with the nuclease-insoluble with the abundance of ORC1 in this that ORC1 ORCs 2–5 to nuclear ORC1 from cells by RNA interference and the of ORC2 in nuclear structures. shown in of of ORC1 into cells, ORC1 This reduction in ORC1 is specific to the ORC1 siRNA, specific for the level of ORC1 their proteins to ORC1 from cells, observed an shift of ORC2 from fractions to fractions This the of ORC2 to fractions as The of MCM onto the ORC1–5 and CDC6 observed the temporal formation of the ORC1–5 complex in non-chromatin nuclear structures in G1 phase human cells. is the of this In budding yeast, MCM is onto origin DNA in an and manner to form the In mammalian cells, the loading of MCM onto chromatin in G1 is with the accumulation of ORC1 Y. S. H. Tsurimoto T. Obuse C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Hidaka M. Stillman B. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This that ORC1–5 complex formation may be required for loading MCM onto chromatin. this the of the on MCM The level of in the chromatin by the reduction of ORC1 in cells suggesting that the loading of MCM onto chromatin is by ORCs 2–5 by the ORC1–5 complex. the of CDC6 from cells by MCM Thus, both CDC6 and are necessary for MCM loading onto chromatin in human cells. is that with the association of ORC2 with the the other of ORC1 also the association of CDC6 with nuclease-insoluble fractions. Thus, the association of CDC6 with nuclear structures and the formation of the ORC1–5 complex on nuclear structures are ORC identified in budding yeast as a complex of six six subunit components of ORC have been identified in eukaryotes, including indicating that the of the complex is conserved among (7Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar, PubMed Scopus Google Scholar). complexes have been from yeast, and although the ORC1–5 complex in human cell by with ORC2 in these by the of mass by with the Thus, in human cells may interact with other ORC subunits only at The of for the ORC1–5 complex been for a expression system (21Dhar S.K. Delmolino L. Dutta A. J. Biol. Chem. 2001; 276: 29067-29071Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 22Vashee 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 that is for ORC it been that Drosophila is for the specific of ORC to origin DNA I. D. Botchan M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). Therefore, a role in the of ORC by interaction with the ORC1–5 complex. it been that human a role as a complex in to role in the initiation of replication Stillman B. Science. 2002; PubMed Scopus Google Scholar). studies of behavior during the cell cycle be necessary to understand how human functions in have demonstrated that the appearance of the ORC1–5 complex is cell and regulated by the ORC1 cycle. ORC2 and are in a complex, and are from ORC2 and in the of Thus, human ORC subunits have a dynamic mechanism that is regulated by the level of This that the subunit of ORC in animal cells is from that in budding yeast and that are each of which a role in regulating replication complex formation at origins protein in human cells is the complex, which is found in the chromatin been that ORC proteins to replication origin in budding yeast, yeast, and Drosophila (1Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (997) Google Scholar, I. D. Botchan M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Kelly T.J. Proc. Natl. Acad. Sci. U. S. 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The complex that have observed associates with ORC1 in to the of the origin association and observed chromatin of ORC subunits during the cell cycle, suggest that the complex associates with chromatin by to to origin this is the human ORCs 2–5 are to replication origin and to budding yeast replication origins be by ORC1 and recruited to nuclear structures where replication assemble of the ORC1–5 complex in nuclear structures of MCM to chromatin, which to pre-RC formation MCM is the ORC1–5 complex by the of ORC1 further initiation including the loading of the DNA This mechanism The the of origins to be from among the bound by the complex. Thus, ORC1 may determine the and order of origin by complexes to nuclear structures The is to that origins are only once per cell cycle, by the of ORC1 This is with that at the of cells and that the origin of cells associate with similar nuclease-insoluble nuclear structures in G1 and dissociate from the initiation of replication in S phase G. B. 2001; PubMed Scopus Google Scholar). is known that in yeast and the loading of MCMs onto the complex to the formation of the pre-RC, a of the complex that This the essential components CDC6 and in to an ORC complex (7Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1392) Google Scholar, PubMed Scopus Google Scholar). with that both ORC1 and CDC6 are required for the loading of MCMs onto chromatin in human cells also observed that the of the complex in chromatin fractions is for MCM loading onto chromatin. In yeast, ORCs form a complex, and the accumulation of proteins a to MCM the human complex functions the budding yeast complex, human ORC1 and CDC6 may as In this the level of human ORC1 that it accumulates in with the loading of MCM, ORC1 may ORC functions as CDC6 in budding ORC1 and CDC6 and are from the J. Smith D. K. J.M. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). human CDC6 a of to that of during S phase, in phase and in the G1 phase J. Hidaka M. Stillman B. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). and C. This although both CDC6 and ORC1 are required for MCM loading in human cells, they have temporal of expression and in pre-RC have demonstrated that human ORC components exhibit a dynamic of during the cell cycle. of the that this temporal and in the initiation of replication in human remain to be how ORC specific how are and in dynamic and in the initiation how further protein for example, those including and and is the of ORC1 to origin be necessary to these and to the mechanism by which DNA replication is regulated in animal cells. Masukata and and for of the and Dutta and for and and for for