Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle.
Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the S phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G(1)-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.
Weinreich
M
M
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Liang
C
C
Chen
H H
HH
Stillman
B
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000705:Anaphase; D000818:Animals; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015825:Chromosomes, Fungal / Q000235:genetics*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D016193:G1 Phase; D005816:Genetic Complementation Test; D007700:Kinetics; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051738:Origin Recognition Complex; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D013489:Suppression, Genetic; D014162:Transfection
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051738:Origin Recognition Complex; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases