The Impact of Vitamin B12 Deficiency on Nuclear de novo Thymidylate Biosynthesis

描述（由申请人提供）：在这些研究中，我们将确定维生素B12在调节核新生dTMP生物合成，维持基因组稳定性中的作用，并阐明维生素B12在出生缺陷中的潜在机制。由于缺乏叶酸饮食的Shmt1-/+小鼠在DNA中积累了尿嘧啶，并对神经管缺陷（NTDs）和结肠癌敏感，dTMP生物合成中的细胞核损伤是饮食方式导致基因中叶酸相关病理的基础。最近，SHMT被确定为在DNA复制和修复位点正确组装dTMP生物合成复合体所需的支架蛋白。维生素B12缺乏导致细胞内5-甲基四氢氢酮和尿嘧啶在DNA中的积累升高，并已提出但未证实导致NTDs。这些拟议的研究将确定维生素B12缺乏对新生dTMP生物合成的影响，并确定该途径中的损伤是否会导致维生素B12相关的NTDs。此外，这些研究的发现将通过确定额外的围孕期措施来进一步预防产前叶酸和维生素B12相关病理的发生，为未来的临床实践提供信息，并为制定基于人群的方法提供决策依据，通过强制强化来改善维生素B12状况。要研究的两个相关和重叠的领域是：目的1 . a)确定维生素B12缺乏是否损害细胞核中dTMP的生物合成。这些研究将确定维生素B12缺乏的潜在机制，正如MTR抑制导致5-甲基thf的不可逆积累所模拟的那样，通过以下方式损害核从头dTMP生物合成：1)dTMP合成的多酶复合物的破坏或2)核苷酸生物合成所需的四氢叶酸辅助因子的消耗。b)确定嘧啶核苷dU和U在核DNA中是否以dUTP或dTTP的形式不同地结合。这些研究将在维生素B12缺乏的细胞模型中确定尿嘧啶在核DNA中的起源，并调和目前文献中关于核苷作为dUTP合并的不确定性。目的二世。确定维生素B12缺乏是否会增加新生dTMP生物合成受损小鼠模型中NTDs的发生率。这些研究将通过我们的小鼠新产dTMP生物合成受损模型，确定母体维生素B12缺乏是否通过饮食相互作用基因增加后代NTD发病率。此外，这些研究将确定维生素B12缺乏是否会损害母体和胎儿基因组的完整性。

DESCRIPTION (provided by applicant): In these studies, we will determine the role of vitamin B12 in regulating nuclear de novo dTMP biosynthesis, maintaining genome stability, as well as elucidate the mechanisms underlying vitamin B12 in birth defects. Impairments nuclear in de novo dTMP biosynthesis underlie folate-associated pathologies in a gene by diet manner, as Shmt1-/+ mice fed a folate-deficient diet accumulate uracil in DNA and are sensitized to developing neural tube defects (NTDs) and colon cancer. Recently, SHMT was identified as a scaffolding protein required for proper assembly of the de novo dTMP biosynthesis complex at sites of DNA replication and repair. Vitamin B12 deficiency results in elevated intracellular 5-methylTHF and uracil accumulation in DNA, and has been suggested but not proved to cause NTDs. These proposed studies will determine the impact of a vitamin B12 deficiency on de novo dTMP biosynthesis and establish whether impairments in this pathway contribute to vitamin B12-associated NTDs. Furthermore, the findings from these studies will inform future clinical practice through identification of additional periconceptional measures to further prevent occurrence of prenatal folate - and vitamin B12-associated pathologies, and inform policy-making decisions in the development of population- based approaches to improve vitamin B12 status through mandated fortification. The two related and overlapping areas to be investigated are: Aim I. a) Determine if vitamin B12 deficiency impairs de novo thymidylate (dTMP) biosynthesis in the nucleus. These studies will determine the potential mechanisms by which vitamin B12 deficiency, as modeled by MTR inhibition leading to an irreversible accumulation of 5-methylTHF, impairs nuclear de novo dTMP biosynthesis via the: 1) disruption of the multi-enzyme complex for dTMP synthesis or 2) depletion of tetrahydrofolate cofactors required for nucleotide biosynthesis. b) Determine if pyrimidine nucleosides, dU and U, are differentially incorporated as either dUTP or dTTP in nuclear DNA. These studies will establish the origin(s) of uracil in nuclear DNA in a cell model of vitamin B12 deficiency, and reconcile the current uncertainty in the literature with respect to the nucleosides that are incorporated as dUTP. Aim II. Determine if vitamin B12 deficiency increases the incidence of NTDs in mouse models of impaired de novo dTMP biosynthesis. These studies will determine if maternal vitamin B12 deficiency increases NTD incidence in offspring through a gene by diet interaction using our mouse model of impaired de novo dTMP biosynthesis. Additionally, these studies will determine if vitamin B12 deficiency compromises genome integrity in maternal and fetal genomes.