ALKYLATING AGENTS: DNA DAMAGE, REPAIR AND MUTAGENESIS

拟议研究的总体目标是了解 烷基化试剂对DNA损伤的分子机制 例如N-甲基-N‘-硝基-N-亚硝胺(MNNG)， N-乙基-N‘-硝基-N-亚硝胺(ENNG)、N-甲基-亚硝脲(MNU)和 N-乙基亚硝脲(ENU)是在真核生物酵母中修复的 酿酒酵母。我们也想了解一下 突变是由这些烷基化试剂引起的。为此，我们 将使用高效液相色谱(HPLC)技术来 核内和线粒体DNA中的修饰碱基的定量 用MNNG、ENNG、MNU和ENU处理细胞后的酵母。我们会 测定核中烷基化产物的脱除量 线粒体DNA作为处理细胞孵育时间的函数， 在使用生长介质的生长条件下和非生长条件下 使用缓冲液的条件，并确定细胞阶段的影响 核DNA的诱变和脱除修饰碱基的循环。我们 将分离和鉴定对MNU和MNU具有超突变能力的突变体 Enu.我们将提纯和鉴定去除O6的酶的活性 来自野生型细胞DNA的烷基鸟嘌呤和可高度突变的突变体 烷基化试剂。乙基化与甲基化的配对研究 BASE将指明修复这些病毒的特异性程度 两种类型的损伤。由于我们的环境中含有许多物质， 是否具有致突变性、致癌性或潜在致突变性和/或致癌性 用于人类的制剂，这些制剂已知会破坏DNA，这是 极其重要的是，我们要描述和理解这些过程 存在于修复DNA损伤的真核生物中。 众所周知，无法修复DNA损伤之间存在相关性 和癌症的诱因，既然也有很好的相关性 在一种物质的致癌性和它的致突变性之间， 我们在了解代理人如何导致突变以及如何导致突变时获得的知识 可修饰的突变诱导将对理解 这些物质是如何致癌的。

The overall objectives of the proposed research are to understand the molecular mechanisms by which damage induced in DNA by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-ethyl-N'-nitro'N-nitrosoguanidine (ENNG), N-methyl-nitrosourea (MNU), and N-ethylnitrosourea (ENU) is repaired in the eucaryotic organism, the yeast Saccharomyces cerevisiae. We would also like to understand the way mutations are induced by these alkylating agents. For this purpose, we will use techniques of high performance liquid chromatography (HPLC) to quantitate the modified bases found in nuclear and mitochondrial DNA of yeast following treatment of cells with MNNG, ENNG, MNU and ENU. We will determine the removal of various alkylation products in nuclear and mitochondrial DNA as a function of time of incubation of treated cells, both under growing conditions using growth medium and non-growing conditions using buffer, and determine the effect of the phase of the cell cycle on mutagenesis and removal of modified bases from nuclear DNA. We will isolate and characterize mutants that are hypermutable to MNU and ENU. We will purify and characterize the enzyme activity that removes O6 alkylguanine from DNA from wild type cells and mutants hypermutable to alkylating agents. Studies on the rapair of the ethylated vs. methylated bases will indicate what degree of specificity exists for repairing these two types of lesions. Since our environment contains many substances which are mutagenic, carcinogenic, or potentially mutagenic and/or carcinogenic agents for humans, and these agents are known to damage DNA, it is extremely important that we characterize and understand the processes available in eucaryotic organisms which exist to repair DNA damage. Correlations are known to exist between the inability to repair DNA damage and the induction of cancer, and since there is also a good correlation between the carcinogenicity of a substance and its mutagenicity, any knowledge we gain in understanding how agents cause mutations and how mutation induction can be modified will be of importance in understanding how these agents cause cancer.