Redundant and differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells

When human cells enter S-phase, overlapping differential inhibitory mechanisms downregulate the replication licensing factors ORC1, CDC6 and Cdt1. Such regulation prevents re-replication so that deregulation of any individual factor alone would not be expected to induce overt re-replication. However, this has been challenged by the fact that overexpression of Cdt1 or Cdt1+CDC6 causes re-replication in some cancer cell lines. We thought it important to analyze licensing regulations in human non-cancerous cells that are resistant to Cdt1-induced re-replication and examined whether simultaneous deregulation of these licensing factors induces re-replication in two such cell lines, including human fibroblasts immortalized by telomerase. Individual overexpression of either Cdt1, ORC1 or CDC6 induced no detectable re-replication. However, with Cdt1+ORC1 or Cdt1+CDC6, some re-replication was detectable and coexpression of Cdt1+ORC1+CDC6 synergistically acted to give strong re-replication with increased mini-chromosome maintenance (MCM) loading. Coexpression of ORC1+CDC6 was without effect. These results suggest that, although Cdt1 regulation is the key step, differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells. Our findings also show for the first time the importance of ORC1 regulation for prevention of re-replication.

[1]  芳賀 慧.  Efficient immortalization of primary human cells by p16[INK4a]-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT , 2010 .

[2]  Anindya Dutta,et al.  Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication* , 2008, Journal of Biological Chemistry.

[3]  Y. Tatsumi,et al.  Identification of novel human Cdt1-binding proteins by a proteomics approach: proteolytic regulation by APC/CCdh1. , 2007, Molecular biology of the cell.

[4]  T. Chiba,et al.  The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted , 2007, The Journal of cell biology.

[5]  E. Kipreos,et al.  C. elegans CUL-4 Prevents Rereplication by Promoting the Nuclear Export of CDC-6 via a CKI-1-Dependent Pathway , 2007, Current Biology.

[6]  M. Fujita,et al.  Efficient immortalization of primary human cells by p16INK4a‐specific short hairpin RNA or Bmi‐1, combined with introduction of hTERT , 2007, Cancer science.

[7]  R. Kobayashi,et al.  CUL4–DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation , 2006, Nature Cell Biology.

[8]  M. Fujita.  Cdt1 revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells , 2006, Cell Division.

[9]  J. Chen,et al.  A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. , 2006, Molecular cell.

[10]  Y. Tatsumi,et al.  Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability , 2006, Journal of Cell Science.

[11]  M. DePamphilis,et al.  Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis , 2006, Journal of Cell Science.

[12]  Anindya Dutta,et al.  PCNA Is a Cofactor for Cdt1 Degradation by CUL4/DDB1-mediated N-terminal Ubiquitination* , 2006, Journal of Biological Chemistry.

[13]  K. Nakayama,et al.  Two E3 ubiquitin ligases, SCF‐Skp2 and DDB1‐Cul4, target human Cdt1 for proteolysis , 2006, The EMBO journal.

[14]  Y. Xiong,et al.  An Evolutionarily Conserved Function of Proliferating Cell Nuclear Antigen for Cdt1 Degradation by the Cul4-Ddb1 Ubiquitin Ligase in Response to DNA Damage* , 2006, Journal of Biological Chemistry.

[15]  J. Walter,et al.  PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication , 2006, Nature Cell Biology.

[16]  J. Diffley,et al.  CDKs Promote DNA Replication Origin Licensing in Human Cells by Protecting Cdc6 from APC/C-Dependent Proteolysis , 2005, Cell.

[17]  D. Takeda,et al.  Degradation of Cdt1 during S Phase Is Skp2-independent and Is Required for Efficient Progression of Mammalian Cells through S Phase* , 2005, Journal of Biological Chemistry.

[18]  J. Blow,et al.  Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re‐replication in Xenopus , 2005, The EMBO journal.

[19]  M. Méchali,et al.  Recombinant Cdt1 Induces Rereplication of G2 Nuclei in Xenopus Egg Extracts , 2005, Current Biology.

[20]  Kazumasa Yoshida,et al.  Intrinsic nuclear import activity of geminin is essential to prevent re‐initiation of DNA replication in Xenopus eggs , 2004, Genes to cells : devoted to molecular & cellular mechanisms.

[21]  J. Walter,et al.  Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts. , 2005, Genes & development.

[22]  B. Calvi,et al.  Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2 , 2004, Development.

[23]  J. Diffley,et al.  Regulation of Early Events in Chromosome Replication , 2004, Current Biology.

[24]  Yunje Cho,et al.  Structural basis for inhibition of the replication licensing factor Cdt1 by geminin , 2004, Nature.

[25]  T. Nishimoto,et al.  Proteolysis of DNA Replication Licensing Factor Cdt1 in S-phase Is Performed Independently of Geminin through Its N-terminal Region* , 2004, Journal of Biological Chemistry.

[26]  Y. Tatsumi,et al.  Cdt1 Phosphorylation by Cyclin A-dependent Kinases Negatively Regulates Its Function without Affecting Geminin Binding* , 2004, Journal of Biological Chemistry.

[27]  Xiaohua Wu,et al.  Cyclin-dependent Kinases Phosphorylate Human Cdt1 and Induce Its Degradation* , 2004, Journal of Biological Chemistry.

[28]  M. Méchali,et al.  Recombinant Cdt 1 Induces Rereplication of G 2 Nuclei in Xenopus Egg Extracts , 2004 .

[29]  Anindya Dutta,et al.  A p53-dependent checkpoint pathway prevents rereplication. , 2003, Molecular cell.

[30]  M. Weitzman,et al.  Adenovirus oncoproteins inactivate the Mre11–Rad50–NBS1 DNA repair complex , 2002, Nature.

[31]  M. Fujita,et al.  Nuclear Organization of DNA Replication Initiation Proteins in Mammalian Cells* , 2002, The Journal of Biological Chemistry.

[32]  B. Stillman,et al.  Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. , 2002, Molecular cell.

[33]  Anindya Dutta,et al.  DNA replication in eukaryotic cells. , 2002, Annual review of biochemistry.

[34]  Carl Co,et al.  Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms , 2001, Nature.

[35]  M. Méchali,et al.  Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin , 2001, Nature Cell Biology.

[36]  J A Wohlschlegel,et al.  Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. , 2000, Science.

[37]  R. Laskey,et al.  Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle. , 2000, Genes & development.

[38]  S. Harrison,et al.  Inhibition of Eukaryotic DNA Replication by Geminin Binding to Cdt1 , 2000 .

[39]  M. Inagaki,et al.  Cell Cycle Regulation of Human CDC6 Protein , 1999, The Journal of Biological Chemistry.

[40]  T. Hunter,et al.  Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Jiri Bartek,et al.  Phosphorylation of mammalian CDC6 by Cyclin A/CDK2 regulates its subcellular localization , 1999, The EMBO journal.

[42]  M. Kirschner,et al.  Geminin, an Inhibitor of DNA Replication, Is Degraded during Mitosis , 1998, Cell.

[43]  J. Parvin,et al.  Human CDC6/Cdc18 Associates with Orc1 and Cyclin-cdk and Is Selectively Eliminated from the Nucleus at the Onset of S Phase , 1998, Molecular and Cellular Biology.

[44]  P. Jallepalli,et al.  Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation. , 1997, Genes & development.

[45]  L. Drury,et al.  The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast , 1997, The EMBO journal.

[46]  M. Fujita,et al.  In Vivo Interaction of Human MCM Heterohexameric Complexes with Chromatin , 1997, The Journal of Biological Chemistry.

[47]  C. Gilbert,et al.  Construction by gene targeting in human cells of a ‘conditional’ CDC2 mutant that rereplicates its DNA , 1997, Nature Genetics.

[48]  V. Dixit,et al.  Elevated levels of syndecan-1 expression confer potent serum-dependent growth in human 293T cells. , 1995, Cancer research.