Mutagenic hot spots & 3-D structure of damaged DNA

这项研究的长期目标有两个：建立 选定的环境诱变剂的诱变潜力并探索 识别和修复受损DNA的分子基础。 在过去的项目期间取得了良好的进展。 定量方法的发展使我们能够确定 确定的 DNA 加合物的体外和体内诱变特征。 基本原则的出现将有助于分析 由环境诱变剂产生的突变谱。例如，使用 位点特异性技术，我们可以预测核苷酸序列背景 突变热点。我们还发现 DNA 聚合酶不同 就其错误编码潜力而言，彼此之间存在显着差异 某些 DNA 加合物。我们还描述了修复的新途径 大肠杆菌中的氧化 DNA 损伤。 在下一个项目期间，我们将寻求确定致突变性 PhIP 和 8-氨基鸟嘌呤（一种修饰的）在体外和体内的潜力 用 2- 处理的动物肝脏中积累的 DNA 碱基 硝基丙烷。我们将使用我们的实验系统来探索分子 无碱基的碱基取代和缺失突变机制 位点、氨基芴加合物和四种立体异构体衍生自 苯并(a)芘。该项目的一部分致力于 DNA 修复研究 酶；我们建议分离和纯化 Fpg 蛋白的同源物 来自哺乳动物的 MutY 蛋白。最后，我们将研究 Fpg 蛋白衍生的单个锌指与受损的相互作用 脱氧核糖核酸。

The long-range goals of this research are two-fold; to establish the mutagenic potential of selected environmental mutagens and to explore the molecular basis for recognition and repaired of damaged DNA. Excellent progress has been made during the past project period. Quantitative methods have been developed that allowed us to establish in vitro and in vivo the mutagenic profiles of defined DNA adducts. Fundamental principles have emerged that will facilitate analyses of mutational spectra produced by environmental mutagens. For example, using site-specific techniques, we can predict nucleotide sequence contexts of mutational hot spots. We also discovered that DNA polymerases differ strikingly from one another in their miscoding potential with respect to certain DNA adducts. We also describe a new pathway for repair of oxidative DNA damage in E. coli. In the next project period, we will seek to determine the mutagenic potential in vitro and in vivo for PhIP and 8-aminoguanine, a modified DNA base which accumulates in the liver of animals treated with 2- nitropropane. We will use our experimental systems to explore molecular mechanisms of base substitutions and deletion mutagenesis for abasic sites, aminofluorene adducts and four stereoisomers derived from benzo(a)pyrene. Part of this project is devoted to studies of DNA repair enzymes; we propose to isolate and purify homologs of Fpg protein and MutY protein from mammalian sources. Finally, we will study the interaction of a single zinc finger derived from Fpg protein with damaged DNA.