Yasutoshi Tatsumi

Cancer cells in the tumour microenvironment use various mechanisms to evade the immune system, particularly T cell attack1. For example, metabolic reprogramming in the tumour microenvironment and mitochondrial dysfunction in tumour-infiltrating lymphocytes (TILs) impair antitumour immune responses2–4. However, detailed mechanisms of such processes remain unclear. Here we analyse clinical specimens and identify mitochondrial DNA (mtDNA) mutations in TILs that are shared with cancer cells. Moreover, mitochondria with mtDNA mutations from cancer cells are able to transfer to TILs. Typically, mitochondria in TILs readily undergo mitophagy through reactive oxygen species. However, mitochondria transferred from cancer cells do not undergo mitophagy, which we find is due to mitophagy-inhibitory molecules. These molecules attach to mitochondria and together are transferred to TILs, which results in homoplasmic replacement. T cells that acquire mtDNA mutations from cancer cells exhibit metabolic abnormalities and senescence, with defects in effector functions and memory formation. This in turn leads to impaired antitumour immunity both in vitro and in vivo. Accordingly, the presence of an mtDNA mutation in tumour tissue is a poor prognostic factor for immune checkpoint inhibitors in patients with melanoma or non-small-cell lung cancer. These findings reveal a previously unknown mechanism of cancer immune evasion through mitochondrial transfer and can contribute to the development of future cancer immunotherapies. Mitochondria with mutations in their DNA from cancer cells can be transferred to T cells in the tumour microenvironment, which leads to T cell dysfunction and impaired antitumour immunity.

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection. Upon DNA damage, eukaryotic cells exhibit a variety of physiological responses, including cell cycle arrest, activation of DNA repair, and apoptosis. Sets of checkpoint proteins that have been conserved with evolution are rapidly induced to prevent replication or segregation of damaged DNA before repair is completed. Related phosphatidylinositol 3-like kinases, ataxia telangiectasia-mutated (ATM) 1The abbreviations used are: ATM, ataxia telangiectacia-mutated; ATR, ATM-Rad3-related; DSB, DNA double strand breaks; FISH, fluorescence in situ hybridization; HFF, human foreskin fibroblast; HSV, herpes simplex virus; hTERT, human telomerase reverse transcriptase gene; HU, hydroxyurea; IR, ionizing radiation; PBS, phosphate-buffered saline; MRN, complex composed of Mre11, Rad50, and Nbs1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; shRNA, short hairpin RNA; GFP, green fluorescent protein; m.o.i., multiplicity of infection; ACV, acyclovir; PAA, phosphonoacetic acid; BrdUrd, bromodeoxyuridine; p.i., postinfection. and ATM-Rad3-related (ATR), respond to a variety of abnormal DNA structures and initiate signaling cascades leading to a DNA damage checkpoint (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). For example, ATM responds to the presence of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR) (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar). On the other hand, the ATR pathway can be stimulated by hydroxyurea (HU), UV light, and base-damaging agents that interfere with the movement of replication forks (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). The ATR pathway also responds to DSBs but more slowly than ATM (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2645) Google Scholar). A variety of checkpoint proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as p53 (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, 5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar). ATM exists as an inactive dimer in the nucleus but undergoes autophosphorylation at Ser-1981 in response to DSBs and dissociates into active monomers (1Westphal C.H. Curr. Biol. 1997; 7: R789-R792Abstract Full Text Full Text PDF PubMed Google Scholar). ATM phosphorylates Chk2 including Thr-68, followed by Chk2 activation (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 7Matsuoka 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 (694) Google Scholar, 8Melchionna R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar). Chk1 is mainly phosphorylated by ATR in response to UV and HU, leading to a 3–5-fold increase in enzyme activity (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Both Mre11 and Nbs1 are also targets of ATM and possibly ATR (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar, 10Gatei M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar, 12D'Amours D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar). The MRN complex consisting of Mre11, Rad50, and Nbs1 has been proposed to facilitate ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 14Usui T. Ogawa H. Petrini J.H. Mol. Cell. 2001; 7: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 15Williams B.R. Mirzoeva O.K. Morgan W.F. Lin J. Dunnick W. Petrini J.H. Curr. Biol. 2002; 12: 648-653Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) and was recently demonstrated to function upstream of ATM activation as a damage sensor, in addition to acting as an effector of ATM signaling (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar). Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are enveloped double-stranded DNA viruses with genomes of 152 and 155 kbp, respectively (17Roizman B. Knipe D.M. Fields B.N. Knipe D.M. Howley P.M. Griffin D.E. Fields Virology. Fourth Ed. Lippincott Williams & Wilkins, Philadelphia, PA2002: 2399-2459Google Scholar). Upon infection immediate-early gene products are expressed and lead to an ordered cascade of viral early and late gene expression. Viral genome is replicated by viral replication machinery, generating highly branched replication intermediates (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The packaging machinery then cleaves concatemeric DNA to monomeric units, which are packaged into preassembled capsids. In HSV-1, DSBs may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection (19Valyi-Nagy T. Olson S.J. Valyi-Nagy K. Montine T.J. Dermody T.S. Virology. 2000; 278: 309-321Crossref PubMed Scopus (73) Google Scholar, 20Milatovic D. Zhang Y. Olson S.J. Montine K.S. Roberts II, L.J. Morrow J.D. Montine T.J. Dermody T.S. Valyi-Nagy T. J. Neurovirol. 2002; 8: 295-305Crossref PubMed Scopus (47) Google Scholar). DSBs are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization (21Huang K.J. Zemelman B.V. Lehman I.R. J. Biol. Chem. 2002; 277: 21071-21079Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22Wohlrab F. Chatterjee S. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6432-6436Crossref PubMed Scopus Google Scholar). is of to host cells can monitor HSV infection as DNA that HSV infection a cellular DNA damage response on DNA damage sensor MRN complex and phosphorylated ATM are recruited to viral replication recognizing newly synthesized viral DNAs as abnormal DNA structures. foreskin cells and green cells were and in Dulbecco's modified Eagle's with fetal calf ataxia were by of the human telomerase reverse transcriptase gene H. H. Y. T. S. M. K. J. 2002; PubMed Scopus (30) Google Scholar). a with a in the gene were by of gene as H. H. Y. T. S. M. K. J. 2002; PubMed Scopus (30) Google Scholar) to of cells with the gene not and cells were in with and while 293T cells with ATM or were in with and human cells were used for of human and the were on cells for by UV light, a of HSV in a was to UV a at a of for The and the gene Y. T. A. K. M. PubMed Scopus Google Scholar) were Y. A HSV was by of and DNAs into cells and by 293T cells as Y. T. A. K. M. PubMed Scopus Google Scholar). were on of cells at multiplicity of infection 1 at were with was used at a of was used at a of T. A. M. Y. H. N. T. J. PubMed Scopus Google Scholar). The was with the virus and in for the of infection. gene was by of and were and and and were Cell and were and highly for and were were with phosphate-buffered and with 1 1 including a and for on of proteins were on and to with and with an were as A. M. T. K. Y. S. Y. T. J. 2003; PubMed Scopus Google Scholar). in were with and then with T. A. M. Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) a on followed by with at The cells were for 1 with in and at with The cells were with the for at then for 1 with or with or and in was as T. A. M. Y. H. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was used at a of other were at and the at synthesized DNAs were by cells with for 1 to were as and then for with 2 to before and with for For an was in products to a of DNA H. M. H. T. M. K. M. T. J. 1991; PubMed Scopus Google Scholar) were with by and used for of HSV was as and then the were in to in on cells were with in and with a in DNA DNA and and cells were at for then at were at with in for and cells were in and in of Viral or cells were with at an of 1 or and at the by into the and at the was for 1 on and virus were by on cells Y. F. Virology. PubMed Scopus Google Scholar). of 293T silencing ATM, the to ATM were expressed as by 293T cells were with the and cells were in B. of the and of 293T cells be HSV a DNA a cellular DNA damage response was induced upon HSV the phosphorylation of DNA proteins in cells The ATM responds to and pathway can during of the cell has been recently proposed that ATM is present as an inactive and is by autophosphorylation at Ser-1981 DSBs or in the M.B. Nature. 2003; PubMed Scopus Google Scholar). in A and of cell that of the phosphorylated of ATM at Ser-1981 upon and although of ATM infection. The MRN complex consisting of Mre11, Rad50, and Nbs1 has been to as a damage sensor D. Jackson S.P. Nat. Rev. Mol. Cell. Biol. 2002; 3: 317-327Crossref PubMed Scopus (718) Google Scholar, 16Carson C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google ATM activation (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). Activated ATM also phosphorylates Nbs1 M. Young D. Cerosaletti K.M. Desai-Mehta A. Spring K. Kozlov S. Lavin M.F. Gatti R.A. Concannon P. Khanna K. Nat. Genet. 2000; 25: 115-119Crossref PubMed Scopus (410) Google Scholar, 11Wu X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google Scholar). in A and increase in of the of Nbs1 was or with or The of Nbs1 was phosphorylated as by not The of Mre11 HSV the C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). In the presence of ATM is known to on Chk2 R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, Piwnica-Worms H. C.E. 2000; Google Scholar). with also phosphorylation of Chk2 at in and infection A and phosphorylation of p53 at a of ATM was at and on or well with the recently that phosphorylation of p53 at on ATM R.D. J. PubMed Scopus Google Scholar). In phosphorylation of p53 to its and in increase in its 2002; PubMed Scopus Google Scholar, Nat. Rev. Cancer. PubMed Scopus Google Scholar). infection had no effect on of p53 is also with the that the p53 are not in the infection R.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). data that HSV infection cellular DNA damage In was not with UV-inactivated virus can and into the cells J. PubMed Google but of the early viral and viral DNA replication not were cells were with a replication-defective virus Y. T. A. K. M. PubMed Scopus Google Scholar) at an of the ATM response was although the DNA was expressed In the presence of ACV, the ATM response by the HSV infection was not is into viral DNA with and viral DNA The of viral DNA viral DNAs and elicit the ATM DNA damage response P. R. B. 2002; PubMed Scopus Google Scholar). In PAA, a of the viral DNA to the ATM DNA damage at multiplicity of infection although the gene viral early was expressed However, at the DNA damage response was induced to in the presence of not viral DNA replication and viral DNA be by host damage the that viral DNA activation of the DNA damage response upon HSV but the that viral gene the DNA damage be that not ATM but also ATR kinases Chk2 at and its activity R. Chen X.B. Blasina A. McGowan C.H. Nat. Cell Biol. 2000; 2: 762-765Crossref PubMed Scopus (264) Google Scholar, Piwnica-Worms H. C.E. 2000; Google Scholar). of p53 at is also by kinases A. Lee A. A. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, K.M. Williams Y. 1999; PubMed Scopus Google Scholar). HSV infection activate the ATM, ATR, or The ATR responds to DNA replication during (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). can also respond to DSBs the but than In to the Chk2 phosphorylation of Chk1 at is known to be mainly by ATR leading to its activation (9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). Chk1 phosphorylation at in and cells phosphorylation was observed in of cells with a well of the replication induced phosphorylation of on the pathway to be in the cells. that replication with types 1 and 2 activation of ATM DNA damage checkpoint signaling than the ATR pathway that responds to replication and infection cellular DNA damage response, the were with HSV Mre11 and Nbs1 the MRN and ATM in Viral DNA replication occurs at discrete sites in nuclei, replication where viral replication proteins cluster and viral DNAs are The viral DNAs have large concatemeric and and structures (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). ATM was recruited to the viral replication The cells were with to of viral or cellular proteins and then to in A and the replication was in sites in the of cells. HSV infection cellular DNA and viral DNA replication A. Chen M. Knipe D.M. Virology. PubMed Scopus Google Scholar). The sites were with the of newly synthesized viral DNA as by and A and the sites of viral DNA proteins were used as for viral replication DNA damage proteins in infection. in in the cells, ATM phosphorylated at Ser-1981 was found to be to and with viral DNAs in the replication the effect of infection on the of the Mre11 and Nbs1 and in of Mre11 and Nbs1 in the as has been J. H. S. A. K. S. T. Tamai K. K. K. Curr. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cerosaletti K.M. Concannon P. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google indicating that the MRN complex is and retained in the damaged Upon Mre11 and Nbs1 proteins to and in the viral replication by the and the of Mre11 and Nbs1 were in the fluorescence to with a indicating that the Mre11 and Nbs1 proteins not but also retained the of newly synthesized viral DNA. The ATM and MRN complex newly synthesized viral DNA in the replication as abnormal DNA structures and to is that the complex function as a of in of viral genome than as a DNA damage and D.E. J. PubMed Scopus Google Scholar) have that Nbs1 with DNA in replication well with DNA by HSV in Nbs1-deficient but ATM-deficient the of ATM and Nbs1 in DNA damage upon HSV phosphorylation of substrates during infection with the was in cells for ATM or Nbs1 In ATM-deficient cells, the of Nbs1 was of Nbs1 phosphorylation of on Chk2 was not of on p53 was not observed and of p53 were as viral replication as was observed in cells These activation of ATM DNA damage pathway by HSV infection. On the other hand, in Nbs1-deficient cells, infection with the to phosphorylation of substrates for ATM activity as ATM Chk2 Thr-68, and p53 although The data that ATM DNA damage were induced by HSV infection in the of a functional MRN complex but that Nbs1 is for activation of ATM DNA damage the in which ATM and MRN complex in the early of the response has been recently that functional MRN is for ATM activation by DSBs and for activation of (13Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (839) Google Scholar). of Nbs1 at Viral of ATM DNA of MRN at viral replication is on the ATM in radiation of cells in of Nbs1 to the damaged nuclei, with of of MRN at sites of damage of ATM in response to DSBs A. Lee A. A. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar, O.K. Petrini J.H. Mol. Cell. Biol. 2001; 21: PubMed Scopus Google Scholar). cells were with of Nbs1 to viral replication The of Nbs1 with that for the viral replication the sites of viral DNA These that of the MRN complex to viral replication is of ATM activation. to ATM at replication in the of a functional MRN cells were with infection in of to the sites of viral replication the ATM to be in the of a functional MRN DNA by HSV Viral the effect of ATM activation on HSV DNA replication, of ATM gene or into 293T cells a and cell be that in 293T cells p53 activity is by downstream as are not in of ATM but not ATR not was ATM silencing a in phosphorylation of Nbs1 by infection as by on The 293T cells with ATM were then with at or and the of in that the of was and 293T cells at a multiplicity of infection. the that virus viral also HSV infection at the of that ATM DNA damage signaling is not for HSV DNA replication and virus in the present that the DNA damage signaling is by HSV probably replication during viral genome The MRN complex in a pathway with ATM X. Ranganathan V. Weisman D.S. Heine W.F. Ciccone D.N. O'Neill T.B. Crick K.E. Pierce K.A. Lane W.S. Rathbun G. Livingston D.M. Weaver D.T. Nature. 2000; 405: 477-482Crossref PubMed Scopus (373) Google ATM as a damage sensor, in addition to acting as an effector of ATM signaling C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar). of ATM, Mre11, and Nbs1 proteins to HSV replication that damage newly synthesized viral DNAs as abnormal DNA structures. with an in of large viral genomes that are as abnormal DNA structures by the MRN leading to activation of cell cycle T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). a ATM damage response signaling is and Chk2, p53, Nbs1, and ATM are phosphorylated C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). HSV DNA replication products are to be large with branched structures that be generated by with replication events (18Lehman I.R. Boehmer P.E. J. Biol. Chem. 1999; 274: 28059-28062Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, M.D. M. Cell. Full Text PDF PubMed Scopus Google Scholar). viral DNA is an a of structures are including large to in DNA as well as structures and replication A. J. PubMed Google Scholar). present data the that newly synthesized viral DNA is by cellular DNA damage has been that a in the of viral replication intermediates Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Boehmer P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). In repair, the MRN with of other the DNA to for DNA and strand the MRN complex be by ATM, Nbs1 is of the MRN complex and is a for ATM phosphorylation (2Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2161) Google Scholar, M. EMBO 2003; PubMed Scopus Google Scholar). The of MRN has activity that is to facilitate DNA M. EMBO 2003; PubMed Scopus Google Scholar, A. L. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that the recruited MRN complex be in of the viral DNA. be in the phosphorylates Chk2 at and p53 at the ATR targets Chk1 at leading to Chk1 activity (5Kastan M.B. Lim D.S. Nat. Rev. Mol. Cell. Biol. 2000; 1: 179-186Crossref PubMed Scopus (661) Google Scholar, 6Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (874) Google Scholar, 9Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (871) Google Scholar). not phosphorylation of on Chk1 in the present The ATR responds to DNA replication during (3Osborn A.J. Elledge S.J. Zou L. Trends Cell Biol. 2002; 12: 509-516Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Our that HSV infection the ATM DNA damage response ATR signaling as is the with of virus replication infection A. M. Zhang L. N. T. Y. H. Y. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (147) Google Scholar). p.i., cells were the but did not exhibit viral replication with to the in cells, suggesting that HSV DNA replication not in cells in which DNA replication has HSV replication fork of DNA replication, ATR DNA damage checkpoint be HSV replication in or with the of an with activity and Virology. 2000; PubMed Scopus Google Scholar). the complex the MRN signaling ATM and ATR C.T. Schwartz R.A. Stracker T.H. Lilley C.E. Lee D.V. Weitzman M.D. EMBO J. 2003; 22: 6610-6620Crossref PubMed Scopus (421) Google Scholar, T.H. C.T. Weitzman M.D. Nature. 2002; PubMed Scopus Google Scholar). the complex function in the of p53 by of p53 P. T. M. D. P.E. 2001; PubMed Scopus Google Scholar). to have to cell cycle checkpoint signaling the with replication of HSV not the MRN the of Mre11 and Nbs1 to be infection However, the viral immediate-early has been to with and p53 R.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The of p53 by prevent p53 downstream signaling and the of and with of HSV infection. the of checkpoint signaling by HSV be of p53 to prevent of for apoptosis. In HSV has been to gene products by the HSV genome as and that downstream of R. Chen Lee H. L. J. 1999; PubMed Google Scholar, M. J. 1999; PubMed Google Scholar, M. J. 2001; PubMed Scopus Google Scholar). HSV host cellular DNA damage and by multiple In no in viral in 293T cells may be

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 at 2.5 growth was by 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 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 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

The activity of human Cdt1 is negatively regulated by multiple mechanisms. This suggests that Cdt1 deregulation may have a deleterious effect. Indeed, it has been suggested that overexpression of Cdt1 can induce rereplication in cancer cells and that rereplication activates Ataxia-telangiectasia-mutated (ATM) kinase and/or ATM- and Rad3-related (ATR) kinase-dependent checkpoint pathways. In this report, we highlight a new and interesting aspect of Cdt1 deregulation: data from several different systems all strongly indicate that unregulated Cdt1 overexpression at pathophysiological levels can induce chromosomal damage other than rereplication in non-transformed cells. The most important finding in these studies is that deregulated Cdt1 induces chromosomal damage and activation of the ATM-Chk2 DNA damage checkpoint pathway even in quiescent cells. These Cdt1 activities are negatively regulated by cyclin A/Cdks, probably through modification by phosphorylation. Furthermore, we found that deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this would be a new molecular mechanism leading to carcinogenesis.