The deoxy pyrimidine nucleotide metabolism in plant mitochondria

胸苷酸（单磷酸、二磷酸、三磷酸，分别称为dTMP、dTDP、dTTP或简称T）是构成DNA四大主要组成部分之一的所有生物体生存所必需的。虽然细菌中的T生物合成已被很好地表征，但对植物线粒体中的T和嘧啶代谢知之甚少。线粒体DNA （mtDNA）合成的T有四种不同的来源：(1)从脱氧单磷酸尿苷（dUMP）甲基化开始的线粒体生物合成(2)从脱氧三磷酸胞苷（dCTP）脱氨开始的线粒体生物合成(3)线粒体中胸腺嘧啶（dT）的回收(4)胸腺嘧啶酸盐从细胞质转运到线粒体。从拟南芥中，我们发现了一种类似于人类dCMP脱氨酶的新型酶（dCTP脱氨酶），但它更倾向于将dCTP脱氨为dUTP，并且与dCMP作为底物的活性很小。我们发现这种酶位于线粒体中，这表明它可能有助于细胞器中的T生物合成。此外，我们首次对一种将dUMP转化为dTMP的植物（拟南芥）核苷酸甲基转移酶（THY2）进行了生化评估，并证实了其线粒体定位。发现了dCTP脱氨酶的单突变体和胸腺嘧啶激酶（TK1b）线粒体异构体THY2的双突变体。这种双突变体在10%的个体中显示出异常的生长模式，这表明线粒体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大小和mtDNA拷贝数，与核DNA进行比较，表明这些突变体的T代谢发生了改变。本提案概述了一种实验方案，以表征生物合成，回收和运输对线粒体脱氧嘧啶核苷酸池的贡献，采用从生物化学和遗传学到代谢组学的各种方法，使用生化分析，下一代测序，LC/MS分析和CRISPR技术。dCTP脱氨酶消耗dCTP并形成T前体的能力使我们假设该酶额外或替代地有助于维持线粒体中的dNTP平衡，从而使突变频率保持在较低水平。我们计划评估线粒体突变率与最近发表的下一代测序方案检测罕见突变。该实验将证明由于缺乏dCTP脱氨酶而导致的脱氧核苷酸池大小不充分平衡是否会改变突变频率。此外，将评估dCTP脱氨酶在解毒5-甲基dCMP中的可能作用-如先前描述的人类同源物。综上所述，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.