MECHANISMS OF HOTSPOT MUTAGENESIS BY (+)-BPDE

这项研究的目的是揭示基本的 化学结构与致突变性/致癌性之间的关系 活性，并提供深入了解的分子机制， 致癌物质在DNA之后，引起突变并导致癌症。 使用 几个位点特异性加合寡核苷酸，我们将比较如何 DNA加合物的结构、构象和位置影响DNA的构象， DNA聚合酶在体外的保真度、持续合成能力和效率。 我们 也将在体内模型中利用这些寡核苷酸来分析 加合物的这些特征如何影响诱发突变。 这些寡聚体含有两个在体内 热点（+）-BPDE（一种最终致癌代谢物， 苯并[a]芘），并在嘌呤碱基上具有（+）-BPDE修饰 或者在热点处或者在热点附近。 非内收和（-）-BPDE（弱或 非致癌对映异构体）加合低聚物也将制备用于 比较研究 具体目标是：1.研究致突变性 引物延伸反应对位点特异性加合物的影响 来自人类的复制型DNA聚合酶和病变旁路 来自酵母的聚合酶。我们将a）准备特定部位的内收物59- 合并模板。B）确定BPDE加合对细胞的影响。 DNA聚合酶的保真度、持续合成能力和效率。C.比较 BPDE立体化学对DNA聚合酶活性的影响。d）、 研究加合物位置对DNA聚合酶活性的影响。2. 用放射免疫法研究位点特异性加合物的体内致突变作用 哺乳动物细胞中的转染测定。 A.构建若干 真核表达载体，使每个含有整个编码 hprt基因区域和单个位点特异性（+）-或（-）-BPDE 在hprt cDNA序列的热点处或附近加合。B）确定 通过体外诱变， 将这些hprt表达载体导入V-79细胞。我们将分析 稳定转化体中的突变，并将它们与突变进行比较。 从我们早期在V-79细胞中的随机诱变研究中获得。 我们 随后将研究加合位置和手性如何影响 变异形成的结果。

The objective of the proposed research is to reveal fundamental relationships between chemical structure and mutagenic/carcinogenic activity, and to provide insight into the molecular mechanisms by which carcinogens after DNA, cause mutations and result in cancer. Using several site-specifically adducted oligonucleotides, we will compare how the structure, conformation and position of the DNA adduct affect the fidelity, processivity and efficiency of DNA polymerases in vitro. We will also utilize these oligonucleotides in an in vivo model to analyze how these characteristics of an adduct affect the induced mutation. These oligomers contain the exact sequence context of two in vivo hotspots for (+)-BPDE (an ultimate carcinogenic metabolite of benzo[a]pyrene) and have the (+)-BPDE modification on the purine base either at or next to the hotspot. Nonadducted and (-)-BPDE (a weak or noncarcinogenic enantiomer) adducted oligomers will also be prepared for comparative studies. The specific aims are to: 1. Study the mutagenic effects of site-specific adducts by the primer extension reaction with the replicative DNA polymerases from human and the lesion bypass polymerase from yeast. We will a) Prepare site-specifically adducted 59- mer templates. b) Determine the effects of BPDE adduction on the fidelity, processivity and efficiency of DNA polymerases. c) Compare the effects of BPDE stereochemistry on DNA polymerase activities. d) Investigate effects of adduct position on DNA polymerase activities. 2. Study the mutagenic effects of site-specific adducts in vivo by transfection assays in mammalian cells. We will a) Construct several eukaryotic expression vectors so that each contains the entire coding region of the hprt gene and a single site-specific (+)- or (-)-BPDE adduct at or next to a hotspot in the hprt cDNA sequence. b) Determine the mutagenic effect of the site-specific adduct in vivo by transfecting these hprt expression vectors into V-79 cells. We will then analyze mutations in stable transformants and compare them with mutations obtained from our earlier random mutagenesis studies in V-79 cells. We will subsequently examine how the adduct position and chirality affect the outcome of the mutation formation.