Genetic toxicology of purine metabolism

描述（由申请人提供）：考虑到癌症病因学，我们建议采用遗传学和生物分析化学相结合的方法来检验嘌呤代谢缺陷影响致突变DNA病变2'-脱氧黄嘌呤（dX）和2'-脱氧肌苷（dl）的细胞负荷的假设。这个新模型得到了初步数据的支持，它扩展了DNA中核碱基脱氨的机制，包括水解、亚硝化和脱氨酶。拟议的研究需要系统分析影响DNA、RNA和大肠杆菌（一种定义良好的模式生物）核苷酸库中黄嘌呤(X)和次黄嘌呤(I)水平的基因。鉴于嘌呤代谢的保守性，该结果将对人类嘌呤代谢遗传多态性与炎症和其他应激相关的遗传毒性和致癌结果产生影响。研究结果还将作为建立嘌呤代谢与癌症和衰老病理生理联系的预测模型的数据库。目标1：发展量化DNA、RNA和核苷酸池中X和I的方法。提出的研究依赖于敏感的分析方法来量化嘌呤代谢突变体中核酸和核苷酸的脱胺产物。我们建议将我们最近开发的定量DNA中dX和dl的LC/MS方法扩展到核苷酸库和RNA中的碱基病变。目标2：分析影响DNA、RNA和核苷酸池中X和I水平的基因。Aim 1中开发的方法现在将应用于大肠杆菌中嘌呤代谢和DNA修复途径突变以及DNA， RNA和核苷酸库中X和I的相应水平的系统，假设驱动的研究。这项工作需要创造新的大肠杆菌突变体，结果将与生物终点相关，如Aim 3中的细胞死亡和诱变，从而创建一个与遗传学和DNA损伤相关的系统数据库。目标3：定义嘌呤代谢和核碱基脱胺产物DNA含量之间关系的机制基础和后果。Aim 2的结果将与细胞毒性、突变、重组和SOS反应等生物学终点相关，并用于定义DNA中dX和dl增加的机制基础。我们将讨论嘌呤代谢缺陷与对亚硝化和氧化应激的敏感性之间的关系。其他研究探讨了DNA修复在DNA水平中dX和dl的作用。与公共卫生的相关性：成功完成拟议的研究将加强我们对内源性DNA损伤的决定因素的理解，内源性DNA损伤在导致癌症和其他疾病途径上的突变中起作用。

DESCRIPTION (provided by applicant): With implications for cancer etiology, we propose to apply a combination of genetics and bioanalytical chemistry to test the hypothesis that defects in purine metabolism affect the cellular burden of the mutagenic DNA lesions, 2'-deoxyxanthosine (dX) and 2'-deoxyinosine (dl). This novel model is supported by preliminary data and it expands the repertoire of mechanisms for nucleobase deamination in DNA, including hydrolysis, nitrosation and deaminases. The proposed studies entail a systematic analysis of genes affecting the levels of xanthine (X) and hypoxanthine (I) in DNA, RNA and the nucleotide pools of E. coli, a well-defined model organism. Given the conserved nature of purine metabolism, the results will have implications for the genotoxic and carcinogenic outcomes of human genetic polymorphisms in purine metabolism in conjunction with inflammation and other stresses. The results will also serve as a database for creating predictive models linking purine metabolism to the pathophysiology of cancer and aging. The three aims are: Aim 1: Develop methods to quantify X and I in DNA, RNA and nucleotide pools. The proposed studies rely on sensitive analytical methodology to quantify the deamination products in nucleic acids and nucleotides in purine metabolism mutants. We propose to extend our recently developed LC/MS method for quantifying dX and dl in DNA to base lesions in the nucleotide pool and in RNA. Aim 2: Analysis of genes affecting the levels of X and I in DNA, RNA and the nucleotide pools. Methods developed in Aim 1 will now be applied to systematic, hypothesis-driven studies of mutations in purine metabolism and DNA repair pathways in E. coli and the consequent levels of X and I in DNA, RNA and the nucleotide pool. This work entails the creation of new E. coli mutants and the results will be correlated with biological endpoints such as cell death and mutagenesis in Aim 3, thus creating a systematic database relating genetics and DNA damage. Aim 3: Defining the mechanistic basis for and consequences of relationships between purine metabolism and DNA content of nucleobase deamination products. Results from Aim 2 will be correlated with biological endpoints such as cytotoxicity, mutation, recombination and the SOS response, and used to define the mechanistic basis for the observed increase in dX and dl in DNA. We will address the relationship between purine metabolic defects and sensitivity to nitrosative and oxidative stress. Other studies address the role of DNA repair in the DNA levels of dX and dl. Relevance to public health: Successful completion of the proposed studies will enhance our understanding of the determinants of endogenous DNA damage, damage that plays a role in causing mutations on the pathway to cancer and other diseases.