Functions of Cytidine Triphosphate Synthase and Inosine Monophosphate Dehydrogenase in Balancing of Nucleotide Pools and Genome integrity

核苷酸生物合成是一个动态过程，在生长和适应非生物刺激（如寒冷）期间显著上调，以满足核酸合成的需求。（脱氧-）CTP和（脱氧-）GTP的提供在该过程中是关键的，因为这些分子通常在核苷酸中是最低的，并且需要增加以避免核苷酸限制和不平衡。三磷酸胞苷合酶（CTPS）和肌苷酸脱氢酶（IMPDH）的蛋白质家族主要负责提供CTP和GTP。在拟南芥中的五种CTPS异构体中，CTPS 2被鉴定为提供用于叶绿体RNA和DNA合成的CTP的主要参与者。CTPS 2的关键功能而没有其他亚型的原因尚不完全清楚，我们建议对CTPS 2的生化特性进行更深入的表征，并测试不同的互补系，以阐明是否需要不同的表达模式或亚型特异性生化特征。该分析将与我们最近初步表征的两种IMPDH亚型的分析平行进行。有趣的是，IMPDH 2 T-DNA插入系表型模仿CTPS 2的amiRNA突变体，表明这两种同种型对于核苷酸平衡至关重要。在这个项目中，我们将与Claus-Peter Witte（汉诺威大学）的研究小组合作，研究突变体对（脱氧）核苷酸水平的影响，并与Julia Bornhorst（伍珀塔尔大学）的研究小组合作，分析突变体中的DNA损伤（由非生物刺激如紫外线引起的）。此外，我们将测试CTPS和IMPDH异构体的物理相互作用，可能存在允许相应的酶活性的完美的协同调节，如已经观察到的在意大利人。

Nucleotide biosynthesis is a dynamic process and markedly upregulated during periods of growth and acclimation to abiotic stimuli, such as cold, to meet the demand of nucleic acid synthesis. Provision of (deoxy-)CTP and (deoxy-)GTP are key in this process as these molecules are typically lowest among nucleotides and need to be increased to avoid nucleotide limitation and imbalance. The protein families of Cytidintriphosphate Synthases (CTPS) and Inosinmonophosphate Dehydrogenases (IMPDH) are mainly responsible to provide CTP and GTP. Among the five CTPS isoforms in Arabidopsis, CTPS2 was identified as main player for the provision of CTP for chloroplast RNA and DNA synthesis. The reason for the pivotal function of CTPS2 and no other isoform is not fully clear and we propose to do a deeper characterization of the biochemical properties of CTPS2 and test different complementation-lines to unravel whether distinct expression patterns or isoform specific biochemical features are required. This analysis will be done in parallel to the analysis of the two IMPDH isoforms, recently preliminary characterized by us. Interestingly, IMPDH2 T-DNA insertion lines phenocopied amiRNA mutants of CTPS2, indicating that those two isoforms are of central importance for nucleotide balancing. In this project we will cooperate with the groups of Claus-Peter Witte (University of Hannover) to investigate the effect of mutants on (deoxy-)nucleotide levels and Julia Bornhorst (University of Wuppertal) to analyzed DNA damage in mutants (triggert by abiotic stimuli such as UV-light. In addition, we will test for a physical interaction of CTPS and IMPDH isoforms that might exist to allow for perfect coregulation of corresponding enzyme activities, as already observed in mammalians.