MECHANISM OF OXIDATIVE DNA DAMAGE IN MODEL ORGANISMS

内源性氧化剂持续击打需氧细胞的DNA的假说已被广泛接受。人们认为，由此产生的损伤是自发突变和衰老的根本原因。因此，当损伤实际发生在细胞内时，需要定义损伤机制的细节。长期以来的模型认为，超氧化物(O2-)是驱动过氧化氢形成羟基自由基的电子的来源。游离铁将催化该反应。然而，在大肠杆菌中的研究表明，另一种未知的还原剂是电子供体，而O2-实际上通过破坏不稳定的酶[4Fe-4S]簇来提供催化铁。我们的目标是：(1)鉴定大肠杆菌中超氧阴离子(O2-)释放铁的脱水酶。氧气应激细胞的DNA损伤率可能取决于这些酶的丰富程度。(2)确定O2-对真核生物的铁库和DNA损伤是否具有相同的影响。一个特别有趣的问题是，核DNA而不是线粒体DNA是否受到保护，因为它远离O2-来源和不稳定的酶。(3)鉴定控制大肠杆菌DNA氧化损伤速率的未知还原剂。我们设计了遗传和生化实验来验证还原剂是NADH的假设。(4)确定一氧化氮是否通过抑制呼吸而加速DNA损伤。已知的呼吸障碍可能通过迫使还原剂积累来加速损害。(5)量化内源性氧化剂引起的复制阻断病变和“自发”病变的比例。这一目标将需要采用定量的聚合酶链式反应分析。我们的长期目标是确定内源性氧化剂对DNA损伤的机制和影响。此外，通过了解代谢扰动如何影响DNA氧化，我们可能能够预测药物治疗对突变和细胞死亡的影响。

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.