ALKYLATING AGENTS: DNA DAMAGE, REPAIR AND MUTAGENESIS

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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.