CONFORMATIONAL STUDIES OF PAH-ADDUCTED OLIGODEOXYNUCLEOTIDES

本计画将探讨多环芳烃加合物与DNA之溶液构象 低聚物，使用高场NMR光谱，增强了其他 生物物理技术，包括电泳迁移率研究， 荧光方法和晶体学。 这些问题将在 在密码子12和61处的人N-ras原癌基因序列的背景。 选择这些是因为PAH诱导的致癌作用与 在这些序列中形成加合物，导致点突变， 导致致癌基因激活。 具体目标有三个方面。 一是 将探索解释特定PAH诱导突变的结构基础 在这些序列中，正如R.S.劳埃德 我们将研究 基于结构的假设，可以解释特定位点的产量， 腺苷酸N6 10 S（61，2）-苯并[a]芘和Bay区的G突变 RAS 61序列中的苯并[a]蒽反式加合物。 二是 将尝试建立PAH共价结构和加合物之间的连接 DNA中的构象 将确定结构-活性关系 一系列PAH加合物位于编码区的位点特异性 Ras 12和Ras 61。 我们将确定PAH的结构特征 决定了嵌入的倾向。 研究与T. M。和CM。哈里斯 将涉及海湾地区和非海湾地区的奔驰[a]蒽， 不同的诱变反应，正如劳埃德所揭示的。 我们将研究一个 一系列10 R和10 S-反式苯并[a]芘鸟嘌呤N2加合物 ras 12以确定10 S（12，2）加合物是否具有不寻常的 由5-邻位空间位阻导出的结构性质 鸟苷，正如Geacintov小组所预测的那样。 将尝试 使用粘性末端分子使PAH-绑架的低聚物结晶。 三是 了解PAH加合物构象如何调节生物化学过程 加合物，将采用多方面的方法来扩展结构 研究与DNA聚合酶的复合物。 将使用两种NMR方法 检测引物-模板复合物。 在第一个转让的NOE中， 与引物-模板复合物之间的相互作用 监测。 在第二种情况下，由蛋白质组成的引物-模板复合物 将使用亚片段。 NMR将补充时间分辨 使用加合物作为荧光探针测定加合物的荧光。 在复合物的活性部位引起变化。 将尝试 以使PAH加合的引物-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.