MECHANISM OF OXIDATIVE DNA DAMAGE IN MODEL ORGANISMS

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The hypothesis that endogenous oxidants continually batter the DNA of aerobic cells has gained widespread acceptance. It is considered plausible that the resultant damage is the root cause of spontaneous mutagenesis and aging. It is therefore desirable to define details of the mechanism of damage as it actually occurs inside the cell. The long-standing model had proposed that superoxide (O2-) was the source of the electrons that drive the formation of hydroxyl radicals from H2O2. Free iron would catalyze the reaction. However, studies in E. coli have shown that another, unknown reductant is the electron donor, while O2-actually provides the catalytic iron by destroying labile enzymic [4Fe-4S] clusters. Our aims are: (1) To identify the dehydratases in E. coli from which superoxide (O2-) releases the iron. The rate of DNA damage in a O2-stressed cell may depend upon the abundance of such enzymes. (2) To determine whether O2- has the same effect upon iron pools and DNA damage in eukaryotes. A particularly interesting question is whether the nuclear DNA, but not the mitochondrial DNA, is protected by its compartmentalization away from O2- sources and labile enzymes. (3) To identify the unknown reductant that controls the rate of oxidative DNA damage in E. coli. We have designed genetic and biochemical experiments to test the hypothesis that the reductant is NADH. (4) To establish whether nitric oxide accelerates DNA damage by inhibiting respiration. Known respiratory blocks accelerate damage, probably by forcing the accumulation of the reductant. (5) To quantify the fraction of replication-blocking lesions and "spontaneous" that are due to endogenous oxidants. This aim will require the adaptation of quantitative PCR analyses. Our long-term goal is to establish both the mechanism and impact of DNA damage by endogenous oxidants. Further, by understanding how metabolic perturbations affect DNA oxidation, we may be able to anticipate the affects of drug therapies upon mutagenesis and cell death.