Genetic toxicology of purine metabolism

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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.