Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.
Evidence obtained from studies with yeast and Xenopus indicate 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 G(1) 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.
Méndez
J
J
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Stillman
B
B
D000911:Antibodies, Monoclonal / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D008938:Mitosis; D009097:Multienzyme Complexes / Q000187:drug effects; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051738:Origin Recognition Complex; D046988:Proteasome Endopeptidase Complex; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D014452:Ubiquitins / Q000378:metabolism; D029867:Xenopus Proteins
D000911:Antibodies, Monoclonal; C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D007976:Leupeptins; C066470:MCM3 protein, human; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D029867:Xenopus Proteins; C085711:mcm5 protein, S pombe; C491206:mcm6a protein, Xenopus; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C099243:MCM4 protein, human; C103258:MCM6 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde