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

一些目前未知功能的蛋白质被认为与化学或酶“意外”导致的代谢物的消除有关。这些酶在体内的特性给分析带来了挑战，因为涉及的受损代谢物很少，而且它们的检测受到阻碍，特别是受到复杂的植物基质的阻碍。然而，由于这些代谢物中的许多都是非生物胁迫的结果，如高温或干旱，它们的去除机制可能有助于抗逆性，因此非常有意义。我们最近开发了一种高度敏感的核苷酸分析方法，可以检测和定量植物中受损的核苷酸。利用这种方法，我们打算研究有助于去除受损核苷酸的分子机制。我们将全面研究两种不同的代谢途径，所获得的经验将有助于识别在应激条件下发生的新的受损核苷酸。在第一个子项目中，我们将研究肌苷(脱氧)-三磷酸((D)ITP)的去除，它被整合到RNA或DNA中后，会损害翻译或复制的精确度。我们将研究一种将(D)ITP转化为典型代谢物肌苷一磷酸的酶，并观察缺乏该酶的突变株的DNA和RNA的变化。此外，我们将鉴定和鉴定另一种从DNA中去除错误结合的dITP的酶。在另一个子项目中，将研究(脱氧)鸟苷三磷酸形成的氧化产物的完全分解代谢。在这里，我们假设已经以典型代谢物的分解代谢为特征的酶是多功能的，也可以转化为典型代谢物的氧化版本。我们将结合遗传分析、生物化学和代谢物测量来全面表征这种受损核苷酸的代谢，包括DNA修复机制所释放的核苷酸碱基的代谢去向。在这个项目的最后部分，我们将建立不同的技术来丰富植物提取物中受损的核苷酸和核苷，并改进它们的质谱学检测。在这里，将使用同位素标记和生物信息学技术(网络分析)等策略来扩大植物中已知的代谢物的空间，特别是关于核苷酸和核苷。

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.