Analysis of Arabidopsisvenosa4-0supports the role of VENOSA4 in dNTP homeostasis

Abstract

ABSTRACTAn imbalance in the deoxyribonucleoside triphosphate (dNTP) pool caused by an increase or decrease in the levels of any of the four dNTPs leads to increased DNA mutations, overloading DNA repair mechanisms. The human protein SAMHD1 (Sterile alpha motif and histidine-aspartate domain containing protein 1) functions as a dNTPase to maintain the balance of the dNTP pool, as well as in DNA repair. In eukaryotes, the limiting step inde novodNTP synthesis is catalyzed by RIBONUCLEOTIDE REDUCTASE (RNR), which consists of two R1 and two R2 subunits. In Arabidopsis, RNR1 is encoded byCRINKLED LEAVES 8(CLS8) and RNR2 by three paralogous genes, includingTSO2(TSO MEANING ’UGLY’ IN CHINESE 2). In plants, thede novobiosynthesis of purines occurs within the chloroplast, and DOV1 (DIFFERENTIAL DEVELOPMENT OF VASCULAR ASSOCIATED CELLS 1) catalyzes the first step of this pathway. Here, to explore the role ofVENOSA4(VEN4), the most likely Arabidopsis ortholog of humanSAMHD1, we studied theven4-0mutant. The mutant leaf phenotype caused by theven4-0point mutation was stronger than those of T-DNA insertionalven4mutations. Structural predictions suggested that the E249L amino acid substitution in the mutated VEN4-0 protein rigidifies its 3D structure compared to wild-type VEN4. The morphological phenotypes of theven4,cls8, anddov1single mutants were similar, and those of theven4 tso2andven4 dov1double mutants were synergistic. Theven4-0mutant had reduced levels of four amino acids related to dNTP biosynthesis, including glutamine and glycine, which are precursors in thede novopurine biosynthesis pathway. Finally, despite its annotation in some databases, At5g40290, a paralog ofVEN4, is likely a pseudogene. These observations support the previously proposed role of VEN4 in dNTP metabolism. Our results reveal a high degree of cross-kingdom functional conservation between VEN4 and SAMHD1 in dNTP homeostasis.