MECHANISM OF OXIDATIVE DNA DAMAGE IN MODEL ORGANISMS

模型生物中 DNA 氧化损伤的机制

• 批准号: 2807383
• 负责人: JAMES A. IMLAY
• 金额: $ 9.51万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2807383
• 关键词: DNA damage; Escherichia coli; NAD(H) phosphate; bacterial DNA; bacterial genetics; bacterial proteins; cytochrome oxidase; eukaryote; hydro lyase; hydrogen peroxide; hydroxyl radical; iron; microorganism culture; nicotinamide adenine dinucleotide; nitric oxide; oxidative stress; oxidizing agents; polymerase chain reaction; respiratory enzyme; superoxides; tissue /cell culture; yeasts

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.

内源性氧化剂不断破坏需氧细胞 DNA 的假说已获得广泛接受。 由此产生的损伤可能是自发突变和衰老的根本原因。 因此，需要定义细胞内部实际发生的损伤机制的细节。 长期存在的模型提出，超氧化物 (O2-) 是驱动 H2O2 形成羟基自由基的电子来源。 游离铁会催化该反应。 然而，对大肠杆菌的研究表明，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) 鉴定大肠杆菌中的脱水酶，超氧化物 (O2-) 可以从中释放铁。 氧气胁迫细胞中 DNA 损伤的速度可能取决于此类酶的丰度。 (2) 确定O2-对真核生物中的铁库和DNA损伤是否具有相同的作用。一个特别有趣的问题是，核 DNA（而非线粒体 DNA）是否受到其远离 O2 源和不稳定酶的分隔的保护。 (3) 鉴定控制大肠杆菌氧化DNA损伤速率的未知还原剂。 我们设计了遗传和生化实验来检验还原剂是 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。 已知的呼吸阻滞可能会通过迫使还原剂积聚来加速损害。 (5)量化由内源性氧化剂引起的复制阻断损伤和“自发性”损伤的比例。这一目标需要采用定量 PCR 分析。我们的长期目标是确定内源性氧化剂损伤 DNA 的机制和影响。 此外，通过了解代谢扰动如何影响 DNA 氧化，我们或许能够预测药物治疗对突变和细胞死亡的影响。