Origin licensing and p53 status regulate Cdk2 activity during G1

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G1/S transition to prevent rereplication. The strict inhibition of licensing after the G1/S transition means that all origins used in S phase must have been licensed in the preceding G1. Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G1 arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G1 progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells.

[1]  R. Sclafani,et al.  Cell cycle regulation of DNA replication. , 2007, Annual review of genetics.

[2]  J. Swedlow,et al.  Temporal Profiling of the Chromatin Proteome Reveals System-wide Responses to Replication Inhibition , 2008, Current Biology.

[3]  J. Harbour,et al.  Cdk Phosphorylation Triggers Sequential Intramolecular Interactions that Progressively Block Rb Functions as Cells Move through G1 , 1999, Cell.

[4]  R. Weinberg,et al.  Physical interaction of the retinoblastoma protein with human D cyclins , 1993, Cell.

[5]  J. Julian Blow,et al.  Preventing re-replication of chromosomal DNA , 2005, Nature Reviews Molecular Cell Biology.

[6]  B. Stillman,et al.  Dynamics of pre-replication complex proteins during the cell division cycle. , 2004, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[7]  D O Morgan,et al.  Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. , 1992, The EMBO journal.

[8]  Z. Tu,et al.  Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. , 2003, Cancer research.

[9]  A. Sancar,et al.  Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. , 2004, Annual review of biochemistry.

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

[11]  J. Cook,et al.  Replication licensing promotes cyclin D1 expression and G1 progression in untransformed human cells , 2009, Cell cycle.

[12]  J. Gautier,et al.  Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. , 2000, Molecular cell.

[13]  H. Nishitani,et al.  Control of DNA replication licensing in a cell cycle , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[14]  T. Mizuno,et al.  Mouse Geminin Inhibits Not Only Cdt1-MCM6 Interactions but Also a Novel Intrinsic Cdt1 DNA Binding Activity* , 2002, The Journal of Biological Chemistry.

[15]  J. Nevins,et al.  The Regulated Association of Cdt1 with Minichromosome Maintenance Proteins and Cdc6 in Mammalian Cells* , 2004, Journal of Biological Chemistry.

[16]  N. Mailand,et al.  Rapid destruction of human Cdc25A in response to DNA damage. , 2000, Science.

[17]  J. Bartek,et al.  DNA damage checkpoints: from initiation to recovery or adaptation. , 2007, Current opinion in cell biology.

[18]  Stéphane Larochelle,et al.  Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells. , 2007, Molecular cell.

[19]  H. Hameister,et al.  Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein. , 1995, Journal of cell science.

[20]  Anindya Dutta,et al.  Right Place, Right Time, and Only Once: Replication Initiation in Metazoans , 2005, Cell.

[21]  M. DePamphilis,et al.  Regulating the licensing of DNA replication origins in metazoa. , 2006, Current opinion in cell biology.

[22]  S. Keenan,et al.  Cyclin-dependent Kinase 2 Nucleocytoplasmic Translocation Is Regulated by Extracellular Regulated Kinase* , 2001, The Journal of Biological Chemistry.

[23]  T. Albrecht,et al.  Cyclin E/Cdk2 activity is controlled by different mechanisms in the G0 and G1 phases of the cell cycle. , 1996, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[24]  Bruce Stillman,et al.  Origin recognition and the chromosome cycle , 2005, FEBS letters.

[25]  Nathan H. Lents,et al.  Stimulation of the Raf/MEK/ERK Cascade Is Necessary and Sufficient for Activation and Thr-160 Phosphorylation of a Nuclear-targeted CDK2* , 2002, The Journal of Biological Chemistry.

[26]  Richard S. Paules,et al.  An ATR- and Chk1-Dependent S Checkpoint Inhibits Replicon Initiation following UVC-Induced DNA Damage , 2002, Molecular and Cellular Biology.

[27]  M. Fujita,et al.  hCDC47, a Human Member of the MCM Family , 1996, The Journal of Biological Chemistry.

[28]  K. Sugimoto,et al.  DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period. , 1994, The EMBO journal.

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

[30]  J. Bartek,et al.  Mammalian G 1-and S-phase checkpoints in response to DNA damage , 2022 .

[31]  Jiri Bartek,et al.  Checking on DNA damage in S phase , 2004, Nature Reviews Molecular Cell Biology.

[32]  R. Roberts,et al.  Transforming growth factor-β induces Cdk2 relocalization to the cytoplasm coincident with dephosphorylation of retinoblastoma tumor suppressor protein , 2004, Breast Cancer Research.

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

[34]  David O. Morgan,et al.  Principles of CDK regulation , 1995, Nature.

[35]  J. Bartek,et al.  Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase) , 1994, The Journal of cell biology.

[36]  W. Bresnahan,et al.  Human cytomegalovirus infection results in altered Cdk2 subcellular localization. , 1997, The Journal of general virology.

[37]  David Beach,et al.  p21 is a universal inhibitor of cyclin kinases , 1993, Nature.

[38]  D. Lane,et al.  Cell type-specific responses of human cells to inhibition of replication licensing , 2002, Oncogene.

[39]  Sibylle Mittnacht,et al.  Differential Phosphorylation of the Retinoblastoma Protein by G1/S Cyclin-dependent Kinases* , 1997, The Journal of Biological Chemistry.

[40]  F. Oesch,et al.  Translocation of cdk2 to the nucleus during G1-phase in PDGF-stimulated human fibroblasts. , 1997, Experimental cell research.

[41]  H. Okayama,et al.  Cdc6 Determines Utilization of p21WAF1/CIP1-dependent Damage Checkpoint in S Phase Cells* , 2008, Journal of Biological Chemistry.

[42]  Bruce Stillman,et al.  Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis , 2000, Molecular and Cellular Biology.

[43]  T. Watanabe,et al.  Modulation of TFIIH‐associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II , 2000, Genes to cells : devoted to molecular & cellular mechanisms.

[44]  Robert A. Weinberg,et al.  Functional Inactivation of the Retinoblastoma Protein Requires Sequential Modification by at Least Two Distinct Cyclin-cdk Complexes , 1998, Molecular and Cellular Biology.

[45]  J. Nevins,et al.  Analysis of Cdc6 function in the assembly of mammalian prereplication complexes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  N. Mailand,et al.  The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis , 2001, Nature.

[47]  R. Abraham Cell cycle checkpoint signaling through the ATM and ATR kinases. , 2001, Genes & development.

[48]  A. Attaran,et al.  BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication , 1995, The Journal of cell biology.

[49]  Jamie K Teer,et al.  Acute Reduction of an Origin Recognition Complex (ORC) Subunit in Human Cells Reveals a Requirement of ORC for Cdk2 Activation* , 2005, Journal of Biological Chemistry.