A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

Abstract

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC. The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC. The six-subunit ORC (1Bell S.P. Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.Nature. 1992; 357 (1579162): 128-13410.1038/357128a0Crossref PubMed Scopus (997) Google Scholar) is the initiator protein that binds to replicator sequences to initiate DNA replication in eukaryotes. ORC is essential for DNA replication in yeasts, flies, mice, and most likely humans (2Bell S.P. Kobayashi R. Stillman B. Yeast origin recognition complex functions in transcription silencing and DNA replication.Science. 1993; 262 (8266072): 1844-184910.1126/science.8266072Crossref PubMed Scopus (368) Google Scholar, 3Foss M. McNally F.J. Laurenson P. Rine J. Origin recognition complex (ORC) in transcriptional silencing and DNA replication in S. cerevisiae.Science. 1993; 262 (8266071): 1838-184410.1126/science.8266071Crossref PubMed Scopus (264) Google Scholar, 4Li J.J. Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system.Science. 1993; 262 (8266075): 1870-187410.1126/science.8266075Crossref PubMed Scopus (364) Google Scholar, 5Loo S. Fox C.A. Rine J. Kobayashi R. Stillman B. Bell S. The origin recognition complex in silencing, cell cycle progression, and DNA replication.Mol. Biol. Cell. 1995; 6 (7579692): 741-75610.1091/mbc.6.6.741Crossref PubMed Scopus (180) Google Scholar, 6Micklem G. Rowley A. Harwood J. Nasmyth K. Diffley J.F. 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A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar). the ORC1 or ORC2 proteins undetectable by the of the the that the ORC at levels the of of that if these ORC2 at to the origins of replication that firing cell cycle that to be an N terminally deleted ORC2 from an internal be at and in the of MCM2–7 as part of the ORC the ORC ring be by CDC6 or ORC of the ORC2 or ORC1 we the by the initiator methionine of ORC5 in HCT116 colon cancer cells and for cells. we ORC5 including in we that if an of the is to initiate from an internal the protein would most of the AAA+ ATPase of ORC5, including the Walker A essential for ATP Even mutation of both of ORC2 in the ORC5 mutant cells viable cell lines that normal DNA replication of ORC The the that either a crippled ORC ORC in the ATPase ring can recruit MCM2–7 to DNA or that human cancer cells can be selected that the of ORC for MCM2–7 recruitment and replication The methionine in the ORC5 is in and is the initiator methionine for the methionine is the internal methionine is at from the Walker A to ATP In if a protein is from methionine deleted most of the AAA+ ATPase of ORC5, a that is for with the AAA+ ATPase in the ORC ring F. Botchan M.R. Berger J.M. Crystal structure of the eukaryotic origin recognition complex.Nature. 2015; 519 (25762138): 321-32610.1038/nature14239Crossref PubMed Scopus (97) Google Scholar). CRISPR-Cas9–mediated DNA at the ATG of ORC5 in HCT116 cells and cell by for ORC5 where ORC5 protein by the around the ATG and mutation in the ORC5 that deleted the that an of ORC5 in these cells, the using and 8 from and 24 and The sequences of these from DNA or and of ORC5 from that in the is by the in ORC5 the of ORC5 ORC5 ORC5 protein from the second methionine at we or ORC5 protein and that both of ORC5 protein by the ORC5 with ORC5 that and or N terminally deleted ORC5 A with of the cell that the can WT of ORC5 in 6 of cell protein in of from cells if ORC5 protein is in these is at the WT of ORC5 in the cells, we the purified by with and we of the protein of We that the from HCT116 cells is similar to that from of a of for and we can that of ORC5 present cell in WT HCT116 cells, that of WT is of that if a deleted ORC5 is in the cells at from an of the would to for and the is to be The of the ORC in the ORC ring is ORC3, ORC5, and ORC4 and ORC5 proteins with in a complex L. A. of the human origin recognition Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and to in the ORC ring F. Botchan M.R. Berger J.M. Crystal structure of the eukaryotic origin recognition complex.Nature. 2015; 519 (25762138): 321-32610.1038/nature14239Crossref PubMed Scopus (97) Google Scholar). The of ORC5 is by the of ORC4 protein the ORC1 to be in with ORC5, is also of the of the ORC3, is in with ORC5, and ORC2 in the cell with the in ORC1 or ORC2 E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google the mutation in ORC5 the levels of ORC6, or CDT1 in cell or the levels of or from the MCM2–7 ring in the of cells to the of these proteins on and the is the The of the ORC subunits, in WT cells, is with the chromatin In the cells, ORC5 and ORC4 proteins on the chromatin and with the chromatin ORC2 and ORC1 on the chromatin the six-subunit ORC to for of the human to be on the chromatin, a with E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google and at with the in the in the six-subunit on the chromatin as S. J. Bell S.P. of origin licensing helicase 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Diffley J.F.X. eukaryotic DNA replication is by helicase 2017; 357 PubMed Scopus Google Scholar). Even the of ORC in CDC6 and we of the chromatin of or in the cells. ORC2 all of the core for to the a core complex E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar). In in the cells, the of ORC3, and to at of the of the core ORC with in the absence of of ORC2 with in cells that at ORC can in the absence of ORC5 in the yeasts, we and reported that ORC1 and with the core in WT human cells and the of with ORC2 A. and of the human Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. of the human origin recognition Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. V. F. and of complex proteins in yeast and mouse Mol. Biol. PubMed Scopus Google Scholar). The of and 24 to that of WT cells, that of enough to the The of 24 or or with WT cells in and a in DNA replication a in the we the of and an of of and in of is as is the of the The show of of or We that the is in cells. with when we the of by cells from an with in 24 cells in the same by WT cells The by a in 24 and for with we in the cells without detectable ORC2 or ORC1 E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar). Cell and DNA replication as as would be if the six-subunit ORC essential for DNA replication in the cell cells with for of cells by DNA origin firing in or DNA a with for by for and for and in DNA WT and cells in number of origins and in the ORC5-depleted cells, we to an in and an of to for the origins. of DNA by cells for with by with in 24 that origins and in the in the cells with ORC5 also in to be in the origins that cell E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar) the cells ORC5 the cells on as we in or cells, we CDC6 by and with WT cells, cell of cells of CDC6 is that CDC6 when the ORC5 is deleted we that cells in a ORC or is by mutation can and E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google and ORC can be deleted in a cancer cell we deleted ORC2 in the cells. or ORC4 proteins ORC2 or ORC5 for and the ORC complex we that of the ORC ring undetectable in these cells. We used to in both of of ORC2 in ORC2 cells, cells in and by to where ORC2 and ORC5 proteins We that the ORC2 ORC5 protein by by an the part of that a of ORC2 protein is in these In we that the of of ORC2 protein at that if ORC2 protein at levels the In to we also the mutation of ORC2 by the DNA and the of ORC2 of WT cells that the of the ORC with the chromatin In to the and subunits, ORC1 also undetectable on the chromatin in to in the ORC5-depleted cells, and on chromatin in of ORC2 the chromatin of MCM2–7 complex E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar). The to recruit MCM2–7, and in the with the in ORC1, or ORC5, and CDT1 with chromatin in the double cells. CDC6 with chromatin in the double cells for with that ORC cells to CDC6 E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar) the in CDC6 with chromatin in the E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar) and cells that CDC6 for the of the ORC complex in cells WT or the cells and viable and and by that both show of cells in the mutation of ORC2 in cells the of cells in by double and to cell in and show a similar of that the cell in that is of the of a of as can be in a and we that the cell in either of the double is of the of we a DNA to the DNA replication and in the undetectable chromatin of subunits, origins of replication and the and origins and WT cells The second a and the on we that the double these cancer cells can WT levels of origins cell origin licensing and can on at normal if ORC2 and ORC5 proteins undetectable and of the of the ORC ring also detectable on we reported that cancer cell lines and in the absence of detectable levels of of ORC1 and we that the for six-subunit ORC for licensing origins of replication can be in cell We to that that all ORC or that ORC be essential in most for of DNA replication. that to for cancer cell lines that in the absence of ORC1 or the that or an internal produce of ORC1 or ORC2 protein that the of for origin In the present we by and the of ORC5 that the mutant ORC5 produce an ORC5 protein with an AAA+ ATPase domain, at levels ORC5 protein would to initiate from a methionine that is from the Walker A would deleted most of the ATPase of Even if independent of a the show that ORC5 protein is the cells recruit MCM2–7, initiate the same number of origins as WT cells cell E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google and and In the of the cells without ORC5 protein the of the cells without detectable ORC2 protein K. Y. P. B. M. J. A. from of requires human ORC and is by Full Text Full Text PDF PubMed Scopus Google Scholar). ORC5 ORC1 and ORC4 ORC2 ORC1, and and In both ORC6, and CDT1 recruitment to chromatin and MCM2–7 for normal DNA replication. the from in yeast and in that a six-subunit ORC to be present on DNA to the of and that of ORC6, with chromatin independent of the subunits, and that CDC6 or CDT1 can be to chromatin independent of of the ORC is also that the of the chromatin binding for ORC6, or CDT1 is of functions at origins of replication, of as functions independent of origin DNA replication is normal in the cells, MCM2–7 binding to chromatin to ORC2 in the cells ORC5 in the cells. in the cells mutant in ORC is that a crippled ORC lacking ORC5 and ORC4 the or ORC2 and the can recruit the with the reported where of the six of ORC on chromatin and MCM2–7 binding is and WT levels of origins of replication likely that MCM2–7 recruitment the ring or a ORC the E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar) MCM2–7 on chromatin, to origin firing. when ORC2 is deleted in cells that to the of ORC5, MCM2–7 with chromatin is at WT be if ORC5 as a of MCM2–7 that in cells is for MCM2–7 binding to The is that the ORC5 cells to a to ORC for MCM2–7 a that when ORC2 deleted by a ring a that is enough to a DNA double with the around the ring in the the of MCM2–7 CDC6 is to the ORC2 and on the of the on the the ring is believed to with the MCM2–7 ring to the of the MCM2–7 ring can be around the DNA F. Botchan M.R. Berger J.M. Crystal structure of the eukaryotic origin recognition complex.Nature. 2015; 519 (25762138): 321-32610.1038/nature14239Crossref PubMed Scopus (97) Google Scholar, 10Yeeles J.T.P. Deegan T.D. Janska A. Early A. Diffley J.F.X. Regulated eukaryotic DNA replication origin firing with purified proteins.Nature. 2015; 519 (25739503): 431-43510.1038/nature14285Crossref PubMed Scopus (324) Google Scholar, S. J. Bell S.P. of origin licensing helicase 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Z. J. E. Li H. Stillman B. L. of the of human origin recognition complex and ATPase 2017; 6 PubMed Google Scholar, N. Y. Y. Li M. J. Tye B.-K. Gao N. of the eukaryotic MCM complex at 2015; PubMed Scopus Google Scholar). We that of and in the same the ORC be to a ring enough to a DNA double or to with the MCM2–7 ring can be if used in the Even with that the of of the in the cells to enough ORC to load normal levels of MCM2–7 on chromatin by ORC6, of is in from the and in the to a The is that ORC subunits, recruit MCM2–7 to the chromatin, the ORC ring is to open the MCM2–7 ring and load around the DNA. We to human MCM2–7 can be to chromatin without the ATPase of ORC. Unlike the in the where Walker A and ORC1 and ORC4 the of human ORC with Walker A and the Walker A and of the ORC1 to be the essential for the ATPase of ORC in and and ATP binding and hydrolysis is essential for ORC I. Remus D. Botchan M. of mutant and origin recognition Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, J. J. J. of the of human origin recognition complex and Walker A Natl. Acad. Sci. U.S.A. PubMed Scopus (45) Google Scholar, Bell S.P. binding of ATP and origin DNA the ATPase of the origin recognition Full Text Full Text PDF PubMed Scopus Google Scholar). In E. M. Y. S. S. A. of human ORC for DNA replication and 2016; PubMed Scopus Google Scholar) we the cells survive with that ORC1 protein we in the cells that ORC1 and ORC4 proteins undetectable on chromatin, and MCM2–7 is at WT levels to chromatin, the that MCM2–7 can be to chromatin to a replicative helicase without the ATPase of ORC. is with that the ATPase of yeast ORC is for MCM2–7 G. Frigola J. Beuron F. Morris E.P. Diffley J.F.X. Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.Mol. Cell. 2014; 55 (25087873): 666-67710.1016/j.molcel.2014.06.034Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The of in MCM2–7 is the of the ATPase ring as a of the ring-shaped The is that the ATPase of the open the MCM2–7 ring to to the DNA. that be essential for human replication is that the MCM ring is in a by CDT1 Y. Cheng E. Wu H. Li N. Yung P.Y.K. Gao N. Tye B.-K. Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer.Nat. Struct. Mol. Biol. 2017; 24 (28191894): 300-30810.1038/nsmb.3374Crossref PubMed Scopus (65) Google Scholar, 15Frigola J. He J. Kinkelin K. Pye V.E. Renault L. Douglas M.E. Remus D. Cherepanov P. Costa A. Diffley J.F.X. Cdt1 stabilizes an open MCM ring for helicase loading.Nat. Commun. 2017; 8 (28643783)1572010.1038/ncomms15720Crossref PubMed Scopus (45) Google Scholar) or is a ring as in where is to DNA by a protein that is to both and R.Y. Abeyrathne P.D. Bell S.D. Mechanism of archaeal MCM helicase recruitment to DNA replication origins.Mol. Cell. 2016; 61 (26725007): 287-29610.1016/j.molcel.2015.12.005Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). the of ORC be to recruit MCM2–7 to sites the and that MCM2–7 can the DNA without the of a also that the recruitment of MCM2–7 to chromatin is on the six-subunit human ORC. Here, of ORC6, and CDT1 be to recruit MCM2–7 to the DNA in the or cells at a that is with that in WT cells. a in the that also the that in the replicative helicase in be on DNA in the absence of the six-subunit ORC. in cells, with a reduction of Asano M. The origin recognition complex is for in Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). of mouse ORC1 that the protein is essential for of cells and for of cells T. Kent L.N. Ouseph M.M. McCarty B. Frank J.J. Kladney R. Cuitino M.C. Thompson J.C. Coppola V. Asano M. Leone G. Endoreduplication of the mouse genome in the absence of ORC1.Genes Dev. 2018; 32 (29967292): 978-99010.1101/gad.311910.118Crossref PubMed Scopus (13) Google Scholar). the to the in the that cells can load enough MCM2–7 on chromatin for replication in the absence of the six ORC. that MCM2–7 recruitment to chromatin and origin firing normal in the cells, for the in cell and the of cells in of ORC also in or K. Stillman B. to and chromosome J. PubMed Scopus Google Scholar, Stillman B. involved in DNA replication, chromosome and PubMed Scopus Google Scholar, K. Stillman B. and number in human PubMed Scopus Google Scholar) would be to these functions for In addition, we functions of as in R. M.M. R. protein recognition protein in and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. chromatin in a Cell Sci. 2016; PubMed Scopus Google Scholar, Z. Stillman B. origin recognition complex is essential for binding to chromatin and Natl. Acad. Sci. U.S.A. 2010; PubMed Scopus (97) Google for the in the normal replication we of ORC cell without replication be in when or for of cells ORC that ORC for cell by for an in replication is In we that the for a six-subunit ORC for licensing origins of replication can be in The of the ORC mutant cells on CDC6 for likely that with ORC6, can the of the ORC ring in recruitment and of the MCM2–7 complex around DNA. HCT116 cells F. A. T. S. J.M. B. for and to DNA PubMed Scopus Google Scholar) in with and The with and with to the CDC6 by the of progression, cells at by double In for and for 8 a second of for Early cells and to in and at HCT116 cells by ORC5 from using and ORC2 using and used ORC5 in cells, ORC5 that is and an using and for and in with by and DNA on at for in and for 8 and in a of or by with either that or that on a and DNA using as previously A. reduction of an origin recognition complex (ORC) in human cells a of ORC for Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) with with for and in DNA in and with and to the with to 24 cells in with Cell and with in cells in The of cells 24 using Cell to the in in and by and with for on in and with used as ORC1 Cell ORC2 ORC3, ORC5, and A. reduction of an origin recognition complex (ORC) in human cells a of ORC for Biol. Full Text Full Text PDF PubMed Scopus Google CDC6 Cell Cell Cell Cell Cell and Cdt1 T. J.C. A. is a for Cdt1 by Biol. Full Text Full Text PDF PubMed Scopus Google ORC5 A. Li Y. J. E. Wu H. in by structure of the of 2015; PubMed Scopus Google Scholar) from with and protein with at the N in in with of for at in and 6 with and with 8 and with as previously J. Stillman B. of human origin recognition complex, and minichromosome maintenance proteins the cell of in Cell Biol. 2000; PubMed Scopus Google Scholar). in A cells for on and at by at in A and in on at and in and in and or to the the We for the cell

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