The deoxy pyrimidine nucleotide metabolism in plant mitochondria

胸苷酸（单，二，三磷酸分别称为dTMP，dTDP，dTTP或短T）是所有生物体的生存所必需的，贡献了DNA的四个主要构建模块之一。虽然T在细菌中的生物合成是相当好的特点，少得多的是已知的T和嘧啶在植物线粒体的代谢。线粒体DNA（mtDNA）合成的T有四种不同来源：（1）从脱氧尿苷一磷酸（dUMP）的甲基化开始的线粒体从头生物合成（2）从脱氧胞苷三磷酸（dCTP）的脱氨基开始的线粒体生物合成（3）线粒体中胸苷（dT）的补救（4）胸苷酸从胞质溶胶转运到线粒体中。从拟南芥中，我们鉴定了一种类似于人dCMP脱氨酶的新型酶（dCTP脱氨酶），但其优选地将dCTP脱氨为dUTP，并且仅对dCMP作为底物具有很小的活性。我们发现，这种酶位于线粒体，这表明它可能有助于T生物合成在这个细胞器。此外，我们第一次生物化学评估的植物（拟南芥）核苷酸甲基转移酶（THY 2）转换dUMP dTMP，并证明其线粒体定位。鉴定了dCTP脱氨酶的单突变体和THY 2与线粒体胸苷激酶亚型（TK 1b）的双突变体。这样的双突变体显示在10%的个人异常的生长模式，表明线粒体T剥夺对植物生理的生理影响。我们将量化线粒体脱氧核苷酸池的大小和mtDNA拷贝数与核DNA相比，表明在这些突变体中T代谢的改变。本提案中概述了一个实验方案，利用生物化学测定、下一代测序、LC/dCTP脱氨酶消耗dCTP并形成T的前体的能力使我们假设该酶另外地或通过CRISPR技术与dCTP脱氨酶结合。或者有助于维持线粒体中的dNTP平衡，从而将突变频率保持在低水平。我们计划用最近发表的检测罕见突变的下一代测序方案来评估线粒体突变率。该实验将证明由缺乏dCTP脱氨酶引起的不充分平衡的脱氧核苷酸池大小是否改变突变频率。此外，dCTP脱氨酶在解毒5-甲基dCMP的可能作用-如前所述的人类同源物-将评估。总之，这些实验将加深我们对线粒体脱氧嘧啶核苷酸代谢及其对线粒体基因组稳定性的影响的理解。

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.