Sequence Effects of AF-modified DNA Structures

描述(由申请人提供)：DNA加合物的结构阐明对于理解致癌的启动和最终设计预防策略至关重要。芳胺类致癌物质与多种人类癌症的病因有关。我们假设：(A)DNA中的芳胺加合物以两种原型构象存在，一种是碱基移位的“堆积”(S)，另一种是外部结合正常的“B型”03)；(B)微妙的S/B构象平衡受加合物部位周围的碱基序列以及复制和修复酶活性部位的特定相互作用性质的调节；以及(C)由此产生的构象异质性在决定突变结果中起着关键作用。我们将使用已有文献记载的由氨基荧烯(AF)诱导的S/B基因的异质性来关联它们进行修复和复制的倾向。这将通过在不同的序列环境中，在有或没有聚合酶存在的情况下，通过使用19F核磁共振与氟标记的AF修饰的DNA相结合来完成。具体目的是：(1)测定不同序列背景下双链或单链/双链双链的S/B比值；(2)测定距病变最近的3‘-侧翼序列发生改变的缺失双链的S/B比值；(3)在聚合酶Klenow片段(KFexo-)的存在下，测定5’-侧翼序列发生变化的18/9-聚模板-引物的S/B比值；以及(4)对AIMS 1和AIMS 3中使用的序列进行定点突变。还将进行圆二色谱和熔融实验，以进一步确定加合物结构。特别是目标3中提出的聚合酶实验，检查了聚合酶活性部位内复制叉处的加合物构象，并可能首次为在模拟生物环境中进行构象和加合物特异性突变提供系统基础。这项拟议的研究将引入19F核磁共振作为一种强大的结构生物学工具来研究芳胺诱变的机制。

DESCRIPTION (provided by applicant): Structural elucidation of DNA adducts is crucial to understanding the initiation of carcinogenesis and ultimately in designing preventative strategies. Arylamine carcinogens are implicated in the etiology of various human cancers. We hypothesize that: (a) arylamine-adducts in DNA exist in two prototype conformations, a base-displaced "stacked" (S) and an external binding normal "B-type" 03); (b) a delicate S/B conformeric balance is modulated by both the base sequence surrounding the adduct site and the nature of specific interactions in the active sites of replication and repair enzymes; and (c) the resulting conformational heterogeneity plays a critical role in determining the mutagenic outcomes. We will use the well-documented aminofluorene (AF)-induced S/B heterogeneity to correlate their propensity to undergo repair and replication. This will be accomplished by the use of 19FNMR coupled with fluorine-labeled AF-modified DNA in various sequence contexts, with or without the presence of a polymerase. Specific aims are to (1) measure the S/B ratios of double or single/double stranded duplexes in various sequence contexts; (2) to measure the S/B ratios of deletion duplexes, whose 3'-next nearest flanking sequence to the lesion is altered; (3) to measure the S/B ratios of 18/9-mer template-primers, whose 5'-flanking sequences are varied, in the presence of the polymerase Klenow Fragment (KFexo-); and (4) to conduct site-specific mutagenesis of the sequences used in Aims 1 and 3. Circular dichroism and melting experiments will also be conducted to define further the adduct structure. The polymerase experiments proposed in Aim 3, in particular, examine the adduct conformation at the replication fork within the active site of a polymerase and may provide,for the first time, a systematic basis for conducting conformation- and adduct-specific mutagenesis in a simulated biological environment. The proposed research will introduce 19F NMR as a powerful structural biology tool in investigating the mechanisms of arylamine mutagenesis.