Genetic toxicology of purine metabolism

描述（由申请人提供）：考虑到癌症病因学的影响，我们建议将遗传学和生物分析化学相结合，以检验嘌呤代谢缺陷影响致突变DNA病变（2 '-脱氧黄嘌呤核苷（dX）和2'-脱氧肌苷（dl））的细胞负荷的假设。这个新的模型得到了初步数据的支持，它扩展了DNA中碱基脱氨的机制，包括水解，亚硝化和脱氨酶。本研究拟对影响大肠杆菌DNA、RNA和核苷酸池中黄嘌呤（X）和次黄嘌呤（I）水平的基因进行系统分析。大肠杆菌，一种定义明确的模式生物。鉴于嘌呤代谢的保守性，研究结果将对嘌呤代谢中人类遗传多态性与炎症和其他应激相关的遗传毒性和致癌性结果产生影响。研究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。目标1：开发定量DNA、RNA和核苷酸库中X和I的方法。拟议的研究依赖于灵敏的分析方法来定量嘌呤代谢突变体中核酸和核苷酸中的脱氨基产物。我们建议将我们最近开发的LC/MS方法用于定量DNA中的DX和DL，以在核苷酸池和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。目的1中开发的方法现在将应用于系统的，假设驱动的研究嘌呤代谢和DNA修复途径在大肠杆菌中的突变。大肠杆菌中的DNA、RNA和核苷酸库中X和I的水平。这项工作需要创建新的E。大肠杆菌突变体，结果将与生物学终点，如细胞死亡和诱变目标3，从而建立一个系统的数据库，遗传学和DNA损伤。目标3：定义嘌呤代谢和核碱基脱氨产物的DNA含量之间关系的机制基础和后果。目标2的结果将与生物学终点（如细胞毒性、突变、重组和SOS反应）相关，并用于定义观察到的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化和氧化应激敏感性之间的关系。其他的研究解决了DNA修复在dX和dl的DNA水平中的作用。与公共卫生的相关性：成功完成拟议的研究将提高我们对内源性DNA损伤的决定因素的理解，这种损伤在导致癌症和其他疾病的途径突变中发挥作用。

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.