CONFORMATIONAL STUDIES OF PAH-ADDUCTED OLIGODEOXYNUCLEOTIDES

该项目将确定加成到DNA上的多环芳烃的溶液构象 低聚物，使用高场核磁共振波谱，由其他物质增强 生物物理技术，包括电泳迁移率研究， 荧光法和结晶学。这些将在 密码子12和61的人类N-ras原癌基因序列的背景。 之所以选择这些，是因为多环芳烃诱导的致癌作用与 在这些序列中形成加合物，导致点突变， 导致癌基因激活。具体目标有三个方面。首先，我们 将探索解释特定多环芳烃诱导突变的结构基础 在这些序列中，如R.S.劳埃德所观察到的。我们将研究 基于结构的假设，可以解释特定部位的A产额到 腺苷N6 10S(61，2)-苯并[a]芘和湾区的G突变 Ras61序列中的苯并[a]菲反式加合物。第二，我们 将尝试建立多环芳烃共价结构和加合物之间的联系 DNA中的构象。将确定结构-活性关系 对于定位于编码区的一系列多环芳烃加合物 RAS12和RAS61。我们将确定PAH的哪些结构特征 决定了夹层的倾向。与T.M.和C.M.哈里斯的研究 将涉及湾区和非湾区的奔驰[a]蒽，它们有 不同的诱变反应，如劳埃德揭示的。我们将研究一个 一系列10R和10S-反式苯并[a]芘在鸟嘌呤氮上的加合物 Ras12确定10S(12，2)加合物是否具有异常 由5-邻的空间位阻衍生的结构性质 鸟苷，正如Geacintov小组预测的那样。我们将尝试 使用粘端分子结晶多环芳烃诱拐的低聚物。第三，到 了解多环芳烃加合物构象如何调节生化过程 对于加合物，将采用多方面的方法来扩展结构 DNA聚合酶复合体的研究。将使用两种核磁共振方法 目的：检测引物-模板复合体。在第一个方面，转移的不 传入的单核苷酸和引物-模板复合体之间将 被监视着。在第二种中，由蛋白质组成的引物-模板复合体 将使用亚片段。核磁共振将补充时间分辨 用加合物作为荧光探针来确定加合物的荧光 诱导该复合体的活性部位发生变化。我们会尝试一次 将多环芳烃加成的引物模板与聚合酶II共结晶。

This project will determine solution conformations of PAHs adducted to DNA oligomers, using high field NMR spectroscopy, augmented by other biophysical techniques, including electrophoretic mobility studies, fluorescence methods, and crystallography. These will be examined in the context of the human N-ras protooncogene sequences at codons 12 and 61. These were chosen because carcinogenesis induced by PAH is correlated with adduct formation in these sequences, leading to point mutations which result in oncogene activation. The specific aims are threefold. First, we will probe for a structural basis to explain specific PAH-induced mutations in these sequences, as observed by R.S. Lloyd. We will examine structurally-based hypotheses which may explain site-specific A yields to G mutations for both adenyl N6 10S(61,2)-benzo[a]pyrene and bay region benz[a]anthracene anti-trans adducts in the ras61 sequence. Second, we will attempt to establish linkage between PAH covalent structure and adduct conformation in DNA. Structure-activity relationships will be determined for a series of PAH adducts located site-specifically in the coding regions of ras12 and ras61. We will determine what structural features of PAH dictate propensity toward intercalation. Studies with T.M. and C.M. Harris will involve both bay- and non-bay-region benz[a]anthracenes, which have different mutagenic responses, as revealed by Lloyd. We will examine a series of 10R and 10S-anti-trans-benzo[a]pyrene adducts at guanine N2 in ras12 to determine whether the 10S(12,2) adduct possesses unusual structural properties derived from steric hindrance of the 5-neighbor guanosine, as predicted by the Geacintov group. An attempt will be made to crystallize PAH-abducted oligomers using sticky ended molecules. Third, to understand how PAH adduct conformation modulates the biochemical processing of adducts, a multifaceted approach will be employed to extend structural studies to complexes with DNA polymerases. Two NMR approaches will be used to examine primer-template complexes. In the first, transferred NOEs between incoming mononucleotides and primer-template complexes will be monitored. In the second, primer-template complexes comprised of protein subfragments will be used. NMR will be supplemented with time-resolved fluorescence using the adduct as a fluorescent probe o ascertain adduct- induced changes at the active site of the complex. An attempt will be made to co-crystallize a PAH-adducted primer-template with polymerase II.