Sequence Effects of AF-modified DNA Structures

描述（由申请人提供）：DNA加合物的结构解析对于理解致癌作用的起始和最终设计预防策略至关重要。芳胺致癌物与各种人类癌症的病因学有关。我们假设：（a）DNA中的芳胺加合物存在两种原型构象，碱基置换的“堆叠”（S）和外部结合的正常“B型”03）;（B）微妙的S/B保形平衡受到加合物位点周围的碱基序列以及复制和修复酶活性位点中特定相互作用的性质的调节;以及（c）所产生的构象异质性在决定诱变结果中起关键作用。我们将使用充分记录的氨基芴（AF）诱导的S/B异质性来关联它们进行修复和复制的倾向。这将通过使用19 FNMR与氟标记的AF修饰的DNA在各种序列环境中偶联来实现，有或没有聚合酶的存在。具体目的是（1）测量不同序列背景下双链或单链/双链双链体的S/B比;（2）测量缺失双链体的S/B比，其离病变的3 '-下一个最近的侧翼序列被改变;（3）测定5 ′-侧翼序列不同的18/9-mer模板引物的S/B比值，在聚合酶Klenow片段（KFexo-）的存在下;和（4）对目的1和3中使用的序列进行位点特异性诱变。还将进行圆二色性和熔融实验以进一步确定加合物结构。特别是在目标3中提出的聚合酶实验，检查聚合酶活性位点内复制叉处的加合物构象，并可能首次提供在模拟生物环境中进行构象和加合物特异性诱变的系统基础。该研究将引入19 F NMR作为研究芳胺诱变机制的强大结构生物学工具。

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.