An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells.
In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.
Hochegger
Helfrid
H
Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto, Japan. helfrid@rg.med.kyoto-u.ac.jp
Dejsuphong
Donniphat
D
Sonoda
Eiichiro
E
Saberi
Alihossein
A
Rajendra
Eeson
E
Kirk
Jane
J
Hunt
Tim
T
Takeda
Shunichi
S
D000483:Alleles; D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D064248:Geminin; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine; D016196:S Phase
D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; C577995:GMNN protein, human; D064248:Geminin; D007976:Leupeptins; D011687:Purines; D000077546:Roscovitine; D016203:CDC2 Protein Kinase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde