Mitochondrial deoxyribonucleoside salvage enzymes : cloning and characterization of deoxyguanosine kinase and thymidine kinase

Mitochondrial deoxyguanosine kinase (dGK) and thymidine kinase (TK2) are responsible for the phosphorylation of deoxyribonucleosides, thus providing DNA precursors for mitochondrial DNA synthesis. Bovine dGK was purified 20,000-fold to apparent homo-geneity. The purified dGK was capable of phosphorylating all the natural occurring purine deoxyribonucleosides, and a number of clinically important anticancer nucleoside analogs, e. g. CdA (2-chloro-2'-deoxyadenosine) and araG (9-p-D-arabinofuranosylguanine). A selective assay method for dGK determination was developed by using araG as substrate.dGK activity levels were elevated in certain brain tumors which may have implications for future anticancer chemotherapy.re anticancer chemotherapy. Amino aicd sequence information from purified bovine dGK was used in RT-PCR to amplify a dGK cDNA fragment. The entire dGK cDNA was subsequently cloned from a human brain cDNA library, and it encoded a 30 kDa protein. The peptide sequences from purified bovine dGK were identified in the deduced amino acid sequence and when expressed in E. coll, the recombinant protein catalysed efficient phosphorylation of dGuo, araG, CdA and dlno similar to purified bovine dGK. Northern blot analysis demonstrated one dominant transcript of 1.35 kb which was found in all tissues at similar levels.A similar approach was used to clone the cDNA for TK2. A TK2 cDNA was cloned from a human brain cDNA library and RACE PCR was used to amplify the cDNA ends from human brain mRNA. All the peptide sequences were found in the deduced amino aicd sequence. Expression of Nterminal truncated TK2 in E. coli yieled a fully active TK2 protein of 25.5 kDa which showed enzymatic properties identical to the purified human and bovine proteins. Northern blot analysis using coding sequence of TK2 cDNA demonstrated several mRNAs, the major ones were 2.2 and 4.0 kb and they were found in most tissues at similar levels.Both dGK and TK2 cDNA sequences show great similarities to the cytosolic deoxycytidine kinase sequence, about 40% at the amino acid level, suggesting that they are evolutionary related and thus established a new enzyme family of mammalian deoxynucleoside kinases.