The deoxy pyrimidine nucleotide metabolism in plant mitochondria

• Applicant: Dr. Marco Herde
• Subject Area: Plant Physiology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 342656586

Thymidylates (mono, di, triphosphates respectively called dTMP, dTDP, dTTP or short T) are indispensable for the survival of all organisms contributing one of the four major building blocks of DNA. While T biosynthesis in bacteria is rather well characterized, much less is known about T and pyrimidine metabolism in plant mitochondria. Four different sources of T for mitochondrial DNA (mtDNA) synthesis can be discerned: (1) de novo biosynthesis in mitochondria starting with the methylation of deoxy uridine monophosphate (dUMP) (2) biosynthesis in mitochondria starting with the deamination of deoxy cytidine triphosphate (dCTP) (3) salvage of thymidine (dT) in mitochondria (4) transport of thymidylates from the cytosol into the mitochondria. From Arabidopsis, we identified a novel enzyme (dCTP deaminase) resembling human dCMP deaminase, but which preferably deaminates dCTP to dUTP and has only little activity with dCMP as substrate. We show that this enzyme is located in mitochondria suggesting that it might contribute to T biosynthesis in this organelle. Additionally, we biochemically assessed for the first time a plant (Arabidopsis) nucleotide methyltransferase (THY2) converting dUMP to dTMP and demonstrated its mitochondrial localization. Single mutants for dCTP deaminase and double mutants for THY2 with the mitochondrial isoform of thymidine kinase (TK1b) were identified. Such double mutants showed in 10% of the individuals an abnormal growth pattern suggesting a physiological impact of mitochondrial T deprivation on plant physiology. We will quantify the mitochondrial deoxy nucleotide pool sizes and the mtDNA copy number in comparison with the nuclear DNA indicating an altered T metabolism in these mutants. An experimental scheme is outlined in this proposal to characterize the contribution of biosynthesis, salvage, and transport to the mitochondrial deoxy pyrimidine nucleotide pools with a variety of approaches spanning from biochemistry and genetics to metabolomics, using biochemical assays, next generation sequencing, LC/MS analyses and CRISPR technology.The ability of dCTP deaminase to consume dCTP and form a precursor of T led us to hypothesize that the enzyme additionally or alternatively contributes to the maintenance of dNTP equilibria in mitochondria thereby keeping the mutation frequency at a low level. We plan to assess the mitochondrial mutation rate with a recently published next generation sequencing protocol detecting rare mutations. This experiment will demonstrate whether insufficiently equilibrated deoxy nucleotide pool sizes caused by the lack of dCTP deaminase alter the mutational frequency. Additionally, a possible role for dCTP deaminase in detoxifying 5-methyl dCMP - as previously described for the human homologue - will be assessed.In summary, these experiments will deepen our understanding of the mitochondrial deoxy pyrimidine nucleotide metabolism and its impact on the stability of the mitochondrial genome.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Claus-Peter Witte