DNA Oxidation Products and Endogenous DNA Adducts

DNA 氧化产物和内源性 DNA 加合物

• 批准号: 6646271
• 负责人: Peter C Dedon
• 金额: $ 31.75万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6646271
• 关键词: DNA; DNA damage; HeLa cells; adduct; amidohydrolases; deoxyribose; high performance liquid chromatography; histones; liquid chromatography mass spectrometry; oxidation; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): The broad hypothesis driving the proposed studies is that the products of deoxyribose oxidation in DNA function as a source of endogenous DNA and protein adducts and thus affect the cellular responses to oxidative stress. The basis for these studies is our observation that, like the lipid peroxidation product malondialdehyde, base propenal derived from deoxyribose 4'-oxidation reacts with dG and DNA to form the mutagenic M1G adduct. The problem is approached with four specific aims: 1) Base propenal and M1G. The contribution of base propenal to the cellular burden of MIG will be assessed in two ways: (1) by quantifying M1G in human cells treated with DNA-directed and non-specific oxidants; (2) by quantifying M1G in model cells containing variable polyunsaturated fatty acid content or [13C]-labeling of deoxyribose in DNA. 2) 3'-Phosphoglycolaldehyde residues and glyoxal adducts. These studies build on our observation that phosphoglycolaldehyde residues react to form glyoxal and its dG adducts. We will extend these studies by (1) comparing the formation of phosphoglycolaldehyde and glyoxal adducts with different oxidizing agents in vitro and in cells; and (2) defining the role of deoxyribose oxidation in the cellular burden of glyoxal adducts. 3) DNA adducts derived from products of deoxyribose 5'-oxidation. Having demonstrated the formation of trans-1,4-dioxo-2-butene in gamma-irradiated DNA, we will quantify this lesion in isolated DNA and cells exposed to different oxidants. We have also shown that cis- and trans-1,4-dioxo-2-butene reacts rapidly with dC to form a novel adduct, so we will develop LC/MS technology to quantify this adduct in cells. Finally, we will investigate the 2-phosphoryl-1,4-dioxobutane residue as a precursor to trans-1,4-dioxo-2-butene. 4) Histone adducts derived from 3'-formylphosphate residues. The reversible acetylation of lysine in histones and other chromatin proteins is recognized as an important control of gene expression. We have obtained evidence consistent with an analogous formylation of histones by formylphosphate residues derived from deoxyribose 5'-oxidation. Given the potential for affecting the physiology of chromatin proteins, we propose to characterize the chemistry and biology of protein formylation in cells by (1) quantifying N6-formyllysine residues in nuclei treated with DNA oxidants; (2) defining the source of N6-formyllysine residues; and (3) characterizing the reaction of histone deacetylases with N6-formyllysine residues.

描述（由申请方提供）：驱动拟定研究的广泛假设是DNA中脱氧核糖氧化产物作为内源性DNA和蛋白质加合物的来源发挥作用，从而影响细胞对氧化应激的反应。这些研究的基础是我们的观察结果，即与脂质过氧化产物丙二醛一样，脱氧核糖4 '-氧化产生的碱性丙烯醛与dG和DNA反应形成致突变性M1 G加合物。这个问题是用四个特定的目标来处理的：1）基地丙烯醛和M1 G。将以两种方式评估碱丙烯醛对细胞内M1 G的贡献：（1）通过定量用DNA定向和非特异性氧化剂处理的人细胞中的M1 G;（2）通过定量含有可变多不饱和脂肪酸含量或DNA中脱氧核糖[13 C]标记的模型细胞中的M1 G。2)3 '-磷酸乙醇醛残基和乙二醛加合物。这些研究建立在我们的观察，磷酸乙醇醛残基反应形成乙二醛和它的dG加合物。我们将通过以下方式扩展这些研究：（1）比较体外和细胞中不同氧化剂对磷酸乙醇醛和乙二醛加合物的形成;（2）确定脱氧核糖氧化在乙二醛加合物细胞负荷中的作用。3)脱氧核糖5 '-氧化产物衍生的DNA加合物。已经证明了反式-1，4-二氧代-2-丁烯在γ-辐射的DNA中的形成，我们将在暴露于不同氧化剂的分离的DNA和细胞中量化这种损伤。我们还表明，顺式和反式-1，4-二氧代-2-丁烯与dC迅速反应形成一种新的加合物，因此我们将开发LC/MS技术来定量细胞中的这种加合物。最后，我们将研究2-磷酰基-1，4-二氧代丁烷残留物作为反式-1，4-二氧代-2-丁烯的前体。4)衍生自3 '-甲酰磷酸残基的组蛋白加合物。组蛋白和其他染色质蛋白中赖氨酸的可逆乙酰化被认为是基因表达的重要控制。我们已经获得了与组蛋白被来自脱氧核糖5 '-氧化的甲酰磷酸残基类似的降解相一致的证据。考虑到影响染色质蛋白质生理学的可能性，我们建议通过以下方式表征细胞中蛋白质脱乙酰化的化学和生物学：（1）定量用DNA氧化剂处理的细胞核中的N6-甲酰赖氨酸残基;（2）定义N6-甲酰赖氨酸残基的来源;和（3）表征组蛋白脱乙酰化酶与N6-甲酰赖氨酸残基的反应。