Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1.
In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation.
Clay-Farrace
Lorena
L
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 2XZ, UK. lc321@cam.ac.uk
Pelizon
Cristina
C
Santamaria
David
D
Pines
Jonathon
J
Laskey
Ronald A
RA
D000470:Alkaloids / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D008938:Mitosis / Q000502:physiology*; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives
D000470:Alkaloids; C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D019311:Staurosporine