DNA Oxidation Products and Endogenous DNA Adducts

描述(申请人提供)：推动这项研究的广泛假设是，DNA中的脱氧核糖氧化产物作为内源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‘-氧化产生的碱基丙二醛与DG和DNA反应生成突变的M1G加合物。这个问题有四个具体的目标：1)基础设施和M1G。碱性普罗普利对MIG细胞负荷的贡献将通过两种方式进行评估：(1)通过量化DNA导向和非特异性氧化剂处理的人类细胞中的M1G；(2)通过量化含有可变多不饱和脂肪酸含量的模型细胞中的M1G或DNA中脱氧核糖的[13C]标记。2)3‘-磷酸乙醛残基和乙二醛加合物。这些研究建立在我们观察到的磷酸二醇醛残基反应生成乙二醛及其DG加合物的基础上。我们将通过(1)比较不同氧化剂在体外和细胞内形成磷酸二醇醛和乙二醛加合物的情况来扩展这些研究；以及(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‘-氧化产物的DNA加合物。在证明了伽马辐射DNA中反式-1，4-二氧代-2-丁烯的形成后，我们将在分离的DNA和暴露于不同氧化剂的细胞中对这种损伤进行量化。我们还发现顺式和反式-1，4-二氧代-2-丁烯与DC快速反应生成一种新的加合物，因此我们将发展LC/MS技术来定量细胞内的这种加合物。最后，我们将研究2-磷酰基-1，4-二氧丁烷残基作为反式-1，4-二氧代-2-丁烯的前体。4)3‘-甲酰基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因素。我们已经获得了与脱氧核糖5‘-氧化产生的甲酰基磷酸残基对组蛋白进行类似甲酰化的证据。考虑到可能影响染色质蛋白质生理的可能性，我们建议通过(1)定量DNA氧化剂处理的细胞核中的N6-甲酰赖氨酸残基；(2)确定N6-甲酰赖氨酸残基的来源；以及(3)表征组蛋白脱乙酰酶与N6-甲酰赖氨酸残基的反应，来表征细胞中蛋白质甲酰化的化学和生物学特征。

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.