Characterization of the molecular network for the maintenance of nucleotide pools in plants

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A number of proteins with currently unknown functions are thought to be involved in the elimination of metabolites resulting from chemical or enzymatic "accidents." The characterization of these enzymes in vivo presents an analytical challenge because the damaged metabolites involved are rare, and their detection is hampered, particularly by a complex plant matrix. However, since many of these metabolites are more abundant as a result of abiotic stresses, such as heat or drought, mechanisms for their removal may contribute to stress resistance and are therefore of great interest.We have recently developed a method for the highly sensitive analysis of nucleotides that allows to detect and quantify damaged nucleotides in plants. Using this method, we intend to investigate the molecular mechanisms that contribute to the removal of damaged nucleotides.Two different metabolic pathways will be investigated comprehensively and the experience gained will help to identify novel damaged nucleotides that occur under stress conditions. In the first subproject, we will investigate the removal of inosine (deoxy)-triphosphate ((d)ITP), which, after incorporation into RNA or DNA, impairs translation or the precision of replication. We will study an enzyme that converts (d)ITP to the canonical metabolite inosine monophosphate and observe changes in DNA and RNA from mutants lacking this enzyme. Furthermore, we will identify and characterize another enzyme that removes erroneously incorporated dITP from DNA. In addition, the metabolic fate of the product from this enzymatic reaction will be investigated.In another subproject, the complete catabolism of an oxidation product formed from (deoxy)guanosine triphosphate will be investigated. Here we hypothesize that enzymes already characterized in the catabolism of canonical metabolites are multifunctional and also convert an oxidized version of the canonical metabolite. We will use a combination of genetic analysis, biochemistry, and metabolite measurements to fully characterize the metabolism of this damaged nucleotide, including the metabolic fate of the nucleobase released as a consequence of DNA repair mechanisms.In the final part of this project, we will establish different techniques to enrich damaged nucleotides and nucleosides in plant extracts and improve their detection by mass spectrometry. Here, strategies such as isotope labeling and bioinformatic techniques (network analysis) will be used to expand the space of metabolites known in plants, especially with respect to nucleotides and nucleosides.