Abstract
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
Highlights
In eukaryotes, responses to DNA damage or replication errors must be coordinated with cell division
Multiple cyclin-dependent kinases (CDKs) control human cell proliferation, but it remains unclear how functions of different CDKs are coordinated during unperturbed cell division or after dividing cells incur DNA damage
We took a chemicalgenetic approach to ask if a specific CDK, Cdk2, played a specialized, non-redundant role in protecting genomic integrity of human cells
Summary
Responses to DNA damage or replication errors must be coordinated with cell division. Checkpoint pathways signal the presence of DNA lesions to the cell-cycle machinery, leading to reversible arrest or apoptosis. The cyclin-dependent kinases (CDKs) that regulate cellcycle progression appear to control aspects of the DNA damage response. CDK activity promotes repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in yeast [1,2,3]. In Saccharomyces cerevisiae, CDK phosphorylates the conserved repair protein Sae to promote DNA-end resection and thereby channel DSBs into the HR pathway [4]. One exception is the Sae homolog CtIP, the phosphorylation of which appears to facilitate resection, and might couple DSB repair pathway choice to cell-cycle position in mammalian cells [7,8]
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