Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana

Abstract

Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/1/145.html) salvage deoxyribonucleosides by transfer of a phosphate group to the 5′ of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/1/21.html)‐like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T‐DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. Database Sequence data from the present study have been deposited in the EMBL database/GenBank under accession numbers: http://www.ebi.ac.uk/gxa/gene/AT3G07800 (AtTK1a), http://www.ebi.ac.uk/gxa/gene/AT5G23070 (AtTK1b) and http://www.ebi.ac.uk/gxa/gene/AT1G72040 (AtdNK).

Role of pyrimidine salvage pathway in the maintenance of organellar and nuclear genome integrity

“…Single homozygous tk1b mutants have been described previously and have been shown to be indistinguishable from the wild type at the adult stage (Clausen et al ., ; Xu et al ., ). At early developmental stages they display smaller yellow cotyledons under normal growth conditions (Xu et al ., ), however, suggesting that chloroplast biogenesis during photomorphogenesis is affected in the absence of TK1b.…”

“…Previous studies have reported that inactivation of both TK genes in Arabidopsis results in a seedling‐lethal phenotype (Clausen et al ., ; Xu et al ., ). To further study the functional redundancy between the two enzymes and to determine whether the addition of sucrose could rescue the double mutants, we crossed the tk1b mutant with the two tk1a mutants characterized previously [SALK_094632, here referred to as tk1a‐1 , as in Clausen et al .…”

Cellular Serpents and Dreaming Spires: New Frontiers in Arginine and Pyrimidine Biology

“…Wolfgang Knecht (Lund University) discussed the diversity of deoxyribonucleoside kinase pyrimidine salvage enzymes across evolution, summarising the different substrate specificities of isoforms in mammals compared to insects and plants (Clausen et al, 2012). He went on to talk about the potential exploitation of these genes in cancer suicide-gene therapy.…”

Extra perspectives of 5-ethynyl-2′-deoxyuridine click reaction with fluorochrome azides to study cell cycle and deoxyribonucleoside metabolism

“…Then, TK functioning in plant cells was questioned [21]; it was assumed that dT phosphorylation was catalyzed by nucleoside phos photransferase (EC 2.7.1.77). Later TK was isolated and purified from broad beans [22], and quite recently two genes encoding TK were identified in the Arabi dopsis genome [23].…”