Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis

ABSTRACT Evidence obtained from studies with yeast and Xenopusindicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G1 and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

[1]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[2]  Bruce Stillman,et al.  ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex , 1992, Nature.

[3]  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.

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

[5]  L. Staszewski,et al.  Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain , 1994, Cell.

[6]  J. Moreau,et al.  XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis , 2000, Nature.

[7]  R. Laskey,et al.  Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication. , 1996, Journal of cell science.

[8]  M. Méchali,et al.  Stepwise Regulated Chromatin Assembly of MCM2–7 Proteins* , 2000, The Journal of Biological Chemistry.

[9]  Mar Sánchez,et al.  The Cdc6 Protein Is Ubiquitinated in Vivo for Proteolysis in Saccharomyces cerevisiae * , 1999, The Journal of Biological Chemistry.

[10]  S. Salghetti,et al.  Destruction of Myc by ubiquitin‐mediated proteolysis: cancer‐associated and transforming mutations stabilize Myc , 1999, The EMBO journal.

[11]  B. Stillman,et al.  A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Laskey,et al.  Human Minichromosome Maintenance Proteins and Human Origin Recognition Complex 2 Protein on Chromatin* , 1998, The Journal of Biological Chemistry.

[13]  G. Williams,et al.  Improved cervical smear assessment using antibodies against proteins that regulate DNA replication. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Diffley,et al.  Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. , 1998, Molecular cell.

[15]  E. Fanning,et al.  The Cdc6 nucleotide-binding site regulates its activity in DNA replication in human cells. , 1999, Molecular biology of the cell.

[16]  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.

[17]  Zoi Lygerou,et al.  The Cdt1 protein is required to license DNA for replication in fission yeast , 2000, Nature.

[18]  Y. Ishimi.  A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex* , 1997, The Journal of Biological Chemistry.

[19]  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.

[20]  M. Baack,et al.  Human Protein MCM6 on HeLa Cell Chromatin* , 1998, The Journal of Biological Chemistry.

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

[22]  D. Gilbert,et al.  Mcm2, but Not Rpa, Is a Component of the Mammalian Early G1-Phase Prereplication Complex , 1999, The Journal of cell biology.

[23]  J. Diffley,et al.  Two steps in the assembly of complexes at yeast replication origins in vivo , 1994, Cell.

[24]  T. Hunt,et al.  The Orc4p and Orc5p Subunits of the Xenopus and Human Origin Recognition Complex Are Related to Orc1p and Cdc6p* , 1998, The Journal of Biological Chemistry.

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

[26]  R V Shohet,et al.  A human protein related to yeast Cdc6p. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[27]  P. Russell,et al.  Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. , 1992, The EMBO journal.

[28]  T. Böhm,et al.  Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. , 1996, Genes & development.

[29]  R. Laskey,et al.  Protein kinase inhibition in G2 causes mammalian Mcm proteins to reassociate with chromatin and restores ability to replicate. , 1998, Experimental cell research.

[30]  L. Staszewski,et al.  Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. , 1994, Cell.

[31]  Anindya Dutta,et al.  Identification of HsORC4, a Member of the Human Origin of Replication Recognition Complex* , 1997, The Journal of Biological Chemistry.

[32]  Andreas Richter,et al.  Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins. , 1996, European journal of biochemistry.

[33]  Bruce Stillman,et al.  Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-Phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase , 2000, Molecular and Cellular Biology.

[34]  Zhen Yan,et al.  PR48, a Novel Regulatory Subunit of Protein Phosphatase 2A, Interacts with Cdc6 and Modulates DNA Replication in Human Cells , 2000, Molecular and Cellular Biology.

[35]  Z. Kelman,et al.  The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[36]  L. Drury,et al.  Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. Nevins,et al.  Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[38]  K. Nasmyth,et al.  S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state , 1995, Current Biology.

[39]  E. Robbins,et al.  Cell synchronization. , 1974, Methods in enzymology.

[40]  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.

[41]  S. Kearsey,et al.  MCM proteins: evolution, properties, and role in DNA replication. , 1998, Biochimica et biophysica acta.

[42]  E V Koonin,et al.  AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. , 1999, Genome research.

[43]  D. Gilbert,et al.  A Distinct G1 Step Required to Specify the Chinese Hamster DHFR Replication Origin , 1996, Science.

[44]  T. Kelly,et al.  cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[45]  S. Forsburg,et al.  The fission yeast cdc18 + gene product couples S phase to START and mitosis , 1993, Cell.

[46]  T. Coleman,et al.  The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts , 1996, Cell.

[47]  J. Diffley,et al.  Uninterrupted MCM2-7 function required for DNA replication fork progression. , 2000, Science.

[48]  B. Stillman,et al.  The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[49]  J. Marquand,et al.  Point of No Return , 1949 .

[50]  P. Nurse,et al.  p65 cdc18 Plays a major role controlling the initiation of DNA replication in fission yeast , 1995, Cell.

[51]  B. Stillman,et al.  Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. , 1997, Genes & development.

[52]  R. Fotedar,et al.  Cell cycle control of DNA replication. , 1995, Progress in cell cycle research.

[53]  Bruce Stillman,et al.  ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome , 1995, Cell.

[54]  M. Bolt,et al.  High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , 1997, Analytical biochemistry.

[55]  L. Drury,et al.  The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle , 2000, Current Biology.

[56]  P. Jallepalli,et al.  Cyclin-dependent kinase and initiation at eukaryotic origins: a replication switch? , 1997, Current opinion in cell biology.

[57]  J. Decaprio,et al.  [7] Cell synchronization , 1995 .

[58]  B. Stillman,et al.  Conserved Initiator Proteins in Eukaryotes , 1995, Science.

[59]  R. Laskey,et al.  Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process. , 2000, Journal of cell science.

[60]  K Nasmyth,et al.  Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. , 1995, The EMBO journal.

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

[62]  K. Stoeber,et al.  Cdc6 protein causes premature entry into S phase in a mammalian cell‐free system , 1998, The EMBO journal.

[63]  P. Jallepalli,et al.  Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe. , 1996, Genes & development.

[64]  O. Aparicio,et al.  Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase , 1997, Cell.

[65]  S. Bell,et al.  Initiation of DNA replication in eukaryotic cells. , 1997, Annual review of cell and developmental biology.

[66]  D. Natale,et al.  Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M–G1 transition in mammals , 2000, The EMBO journal.

[67]  B. Stillman,et al.  Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. , 2000, Genes & development.

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

[69]  E. Harlow,et al.  Using Antibodies: A Laboratory Manual , 1999 .

[70]  Anindya Dutta,et al.  ORC5L, a New Member of the Human Origin Recognition Complex, Is Deleted in Uterine Leiomyomas and Malignant Myeloid Diseases* , 1998, The Journal of Biological Chemistry.

[71]  R. Laskey,et al.  The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells. , 2000, Journal of structural biology.