Abstract
Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.
Highlights
Intracellular concentrations of deoxyribonucleotide triphosphates are tightly regulated to avoid mutagenesis during DNA replication and repair [1]
The functional role of deoxycytidine kinase in DNA repair Our results show that ionizing radiation (IR) induces significant changes in deoxycytidine metabolism by activating dCK and increasing the salvage of deoxycytidine from the extracellular space in L121010K murine leukemia cell line
The importance of ataxia telangiectasia mutated (ATM) in dCK activation that we observe agrees with a recent study by Yang et al who show that ATM phosphorylates dCK at Ser74 after IR [33]
Summary
Intracellular concentrations of deoxyribonucleotide triphosphates (dNTPs) are tightly regulated to avoid mutagenesis during DNA replication and repair [1]. Mammalian cells synthesize dNTPs by two mechanisms: 1) the de novo pathway converts glucose and amino acids to deoxyribonucleotides via ribonucleotide reductase (RNR); 2) the deoxyribonucleoside (dN) salvage pathway generates dNTPs through sequential phosphorylation of recycled deoxyribonucleosides [2]. Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the dN salvage pathway, capable of phosphorylating deoxycytidine (dC), deoxyadenosine (dA) and deoxyguanosine (dG) [3,4]. Phosphorylation of serine 74 (Ser74) was shown to be critical in regulating enzyme activity [18,19,20]. DCK can adopt an open state, capable of substrate binding, or a closed, catalytically active, state [21,22]. Serine to glutamic acid (S74E) substitution mimicking Ser phosphorylation favors the open state and dramatically reduces phosphorylation of purines (dA and dG) but not pyrimidine dC [22]
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