DNA Oxidation Products and Endogenous DNA Adducts

描述（由申请人提供）：推动拟议研究的广泛假设是DNA中脱氧核糖氧化的产物作为内源性DNA和蛋白质加合物的来源，从而影响细胞对氧化应激的反应。这些研究的基础是我们观察到，与脂质过氧化产物丙二醛一样，脱氧核糖4'-氧化衍生的碱基原体与dG和DNA反应形成诱变的M1G加合物。这个问题有四个具体目标：1)基地检察官和M1G。将通过两种方式评估碱基促生酶对MIG细胞负荷的贡献：(1)通过dna定向和非特异性氧化剂处理的人细胞中的M1G定量；(2)通过定量多不饱和脂肪酸含量可变的模型细胞中的M1G或DNA中脱氧核糖的[13C]标记。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观察到磷酸乙醇醛残基反应形成乙二醛及其dG加合物的基础上。我们将扩展这些研究：(1)比较不同氧化剂在体外和细胞中形成磷酸乙醇醛和乙二醛加合物；(2)确定脱氧核糖氧化在乙二醛加合物的细胞负荷中的作用。脱氧核糖5'-氧化产物衍生的DNA加合物。已经证明了反式-1,4-二氧基-2-丁烯在γ辐照DNA中的形成，我们将在分离的DNA和暴露于不同氧化剂的细胞中量化这种损伤。我们还发现顺式和反式-1,4-二氧基-2-丁烯与dC快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-磷酸基-1,4-二氧丁烷残基作为反式-1,4-二氧丁烯的前体。4) 3'-甲酰磷酸残基衍生的组蛋白加合物。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。我们已经获得了与脱氧核糖5'-氧化产生的甲酰磷酸残基类似的组蛋白甲酰化一致的证据。考虑到影响染色质蛋白生理的潜力，我们建议通过(1)定量DNA氧化剂处理过的细胞核中的n6 -甲酰赖氨酸残基来表征细胞中蛋白质甲酰化的化学和生物学特性；(2)确定n6 -甲酰基赖氨酸残基的来源；(3)描述组蛋白去乙酰化酶与n6 -甲酰基赖氨酸残基的反应。

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.