Basis for sequence selective guanine oxidation in DNA

描述(由申请人提供)： 拟议的研究针对炎症的化学介质亚硝基过氧碳酸酯(ONOOCO2-)造成的损伤具有不寻常的DNA序列选择性的基础。与经典的单电子DNA氧化剂相反，观察到DNA中ONOOCO2-对鸟嘌呤(G)的反应性随着计算的序列相关G电离势的增加而增加。相反，G对光氧化的反应性随着电离势的增加而降低。我们建议研究这一现象以检验几个假设：(1)ONOOCO2-的行为对于带负电荷的氧化剂是共同的；(2)这种行为反映了暴露在更多溶剂中的G的优先氧化；以及(3)初始的单电子氧化事件决定了ONOOCO2-和其他氧化剂氧化G的选择性。这些结果将通过揭示DNA损伤和随后突变的位置的化学基础，为炎症和癌症之间的机制联系提供新的见解。目的1：确定碳酸盐自由基阴离子(CO3)、ONOO-、ONOOCO2-及相关氧化剂的DNA序列选择性。我们将扩大初步研究，以包括一系列DNA序列(寡核苷酸、32P-DNA片段和基因组DNA)和ONOOCO2-浓度。我们还将通过比较ONOOCO2-与各种带负电荷、中性和带正电荷的氧化剂的序列选择性来检验G在DNA中的反应活性受氧化剂电荷影响的假设。目的2：确定CO3、ONOO-和ONOOCO2-的序列选择性的基础。首先，我们验证了G氧化的序列选择性是在氧化反应的第一步而不是后来的产物形成的结果的假设。然后，我们继续检验溶剂暴露控制G对ONOOCO2-和其他氧化剂的反应活性的假设：(1)比较单链和双链寡核苷酸的序列选择性；(2)评估G在不同DNA序列背景下的亚米诺质子交换率。目的3：比较DNA损伤化学对CO3的序列选择性。和ONOOCO2-。为了补充目标#1和#2，我们将测试G氧化产物随序列背景而变化的假设。首先，我们定义了经氧化剂处理的质粒DNA中碱基和脱氧核糖的氧化比例，然后继续研究DNA序列背景在G氧化产物谱中的作用。寡核苷酸中的鸟嘌呤损伤将通过LC-MS对单个G序列进行表征，并通过核酸外切/MALDI-MS对具有多个G序列的损伤进行定位。

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.