Basis for sequence selective guanine oxidation in DNA

描述（由申请人提供）： 拟议的研究解决了一个不寻常的DNA序列选择性的基础上产生的损伤亚硝基过氧碳酸盐（ONOOCO 2-），炎症的化学介质。与“经典”单电子DNA氧化剂相反，ONOOCO 2-对DNA中鸟嘌呤（G）的反应性被观察到作为计算的序列依赖性G电离势的函数而增加。相反，G对光氧化的反应性随着电离势的增加而降低。我们建议调查这一现象，以测试几个假设：（1）ONOOCO 2-的行为是常见的带负电荷的氧化剂;（2）的行为反映优先氧化的G的，是更多的溶剂暴露;和（3），最初的单电子氧化事件决定的选择性G氧化ONOOCO 2-和其他氧化剂。这些结果将通过揭示DNA损伤和后续突变位置的化学基础，为炎症和癌症之间的机制联系提供新的见解。目的1：确定碳酸根阴离子（CO 3）对DNA序列的选择性，ONOO-、ONOOCO 2-和相关氧化剂。我们将扩展初步研究，包括一系列DNA序列（寡核苷酸，32 P-DNA片段和基因组DNA）和ONOOCO 2浓度。我们还将测试的假设，即在DNA中的G的反应性是由氧化剂的电荷的影响，通过比较ONOOCO 2-的序列选择性，为各种带负电荷的，中性和带正电荷的氧化剂。目标2：确定CO 3的序列选择性的基础，ONOO-和ONOOCO 2-。首先，我们测试的假设，G氧化的序列选择性出现在氧化反应的第一步，而不是作为后来的产物形成的结果。然后，我们继续测试的假设，即溶剂暴露控制的G的对ONOOCO 2-和其他氧化剂的反应性：（1）比较单链和双链寡核苷酸的序列选择性;和（2）评估的亚氨基质子交换率的G的在不同的DNA序列背景。目的3：比较CO 3对DNA损伤化学的序列选择性。ONOCO2-。为了补充目标#1和#2，我们将检验G氧化产物作为序列背景的函数而变化的假设。首先，我们定义的比例与氧化剂处理的质粒DNA中的碱基和脱氧核糖氧化，然后着手调查的作用，DNA序列的背景下的G氧化产物的光谱。寡核苷酸中的鸟嘌呤损伤将通过LC-MS表征单个G序列，并通过核酸外切酶消化/MALDI-MS表征多个G序列中的损伤。

DESCRIPTION (provided by applicant): The proposed studies address the basis for an unusual DNA sequence selectivity for damage produced by nitrosoperoxycarbonate (ONOOCO2-), a chemical mediator of inflammation. In a manner opposite to "classical" one electron DNA oxidants, the reactivity of ONOOCO2- toward guanine (G) in DNA was observed to increase as a function of the calculated sequence-dependent G ionization potential. In contrast, G reactivity toward photooxidation decreases with increasing ionization potential. We propose to investigate this phenomenon to test several hypotheses: (1) that the behavior of ONOOCO2- is common to negatively-charged oxidants; (2) that the behavior reflects preferential oxidation of G's that are more solvent exposed; and (3) that the initial one-electron oxidation event determines the selectivity of G oxidation by ONOOCO2- and other oxidants. The results will provide new insights into the mechanistic link between inflammation and cancer by revealing the chemical basis for the location of DNA damage and subsequent mutations. Aim 1: Define the DNA sequence selectivity of carbonate radical anion (CO3.), ONOO-, ONOOCO2- and related oxidants. We will extend preliminary studies to include a range of DNA sequences (oligos, 32P-DNA fragments and genomic DNA) and ONOOCO2- concentrations. We will also test the hypothesis that the reactivity of a G in DNA is influenced by the charge of the oxidant by comparing the sequence selectivity of ONOOCO2- to that for a variety of negatively-charged, neutral and positively-charged oxidants. Aim 2: Define the basis for the sequence selectivity of CO3., ONOO- and ONOOCO2-. First, we test the hypothesis that the sequence selectivity of G oxidation arises at the first step of the oxidation reaction and not as a result of later product formation. We then proceed to test the hypothesis that solvent exposure governs the reactivity of G's toward ONOOCO2- and other oxidants by: (1) comparing sequence selectivity in single- and double-stranded oligos; and (2) assessing the imino proton exchange rates of G's in different DNA sequence contexts. Aim 3: Compare the sequence selectivity of DNA damage chemistry for CO3. and ONOOCO2-. To complement Aims #1 and #2, we will test the hypothesis that the products of G oxidation vary as a function of sequence context. First, we define the proportions of base and deoxyribose oxidation in plasmid DNA treated with the oxidants and then proceed to investigate the role of DNA sequence context in the spectrum of G oxidation products. Guanine lesions in the oligos will be characterized by LC-MS for single G sequences and by exonuclease digestion/MALDI-MS for localizing damage in sequences with multiple G's.