Genetic toxicology of purine metabolism

描述(申请人提供)：结合癌症病因学，我们建议应用遗传学和生物分析化学相结合的方法来检验这一假说，即嘌呤代谢缺陷会影响致突变DNA损伤的细胞负荷，即2‘-脱氧黄嘌呤(Dx)和2’-脱氧肌苷(Dl)。这个新的模型得到了初步数据的支持，它扩展了DNA中碱基脱氨的各种机制，包括水解、亚硝化和脱氨酶。拟议的研究需要对影响DNA、RNA中黄嘌呤(X)和次黄嘌呤(I)水平的基因以及大肠杆菌的核苷酸池进行系统分析。鉴于嘌呤代谢的保守性，这一结果将对人类嘌呤代谢基因多态与炎症和其他应激反应的遗传毒性和致癌结果产生影响。这些结果还将作为一个数据库，用于创建将嘌呤代谢与癌症和衰老的病理生理学联系起来的预测模型。三个目标是：目标1：开发量化DNA、RNA和核苷酸池中的X和I的方法。拟议的研究依赖于灵敏的分析方法来定量核酸中的脱氨基产物和嘌呤代谢突变体中的核苷酸。我们建议将我们最近开发的定量DNA中dx和dl的LC/MS方法扩展到核苷酸池和RNA中的碱基损伤。目的2：分析影响DNA、RNA和核苷酸池中X和I水平的基因。目标1中开发的方法现在将应用于系统的、假设驱动的研究，研究大肠杆菌中嘌呤代谢和DNA修复途径的突变，以及由此导致的DNA、RNA和核苷酸池中X和I的水平。这项工作需要创造新的大肠杆菌突变体，结果将与AIM 3中的细胞死亡和突变等生物终点相关，从而创建一个关于遗传学和DNA损伤的系统数据库。目的3：确定核苷酸脱氨基产物的嘌呤代谢和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反应等生物终点相关，并用于确定观察到的DNA中dx和dl增加的机制基础。我们将讨论嘌呤代谢缺陷与亚硝酸盐和氧化应激敏感性之间的关系。其他研究涉及DNA修复在dx和dl DNA水平中的作用。与公众健康相关：拟议研究的成功完成将加强我们对内源性DNA损伤决定因素的了解，内源性DNA损伤在导致癌症和其他疾病的途径上起到突变的作用。

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.