The induction of DNA damages by physical and chemical agents is an early step for mutagenesis, carcinogenesis and cell inactivation by most cancer therapies. Cells have evolved eloquent enzyme systems for recognition and elimination of such damages as a defense against genetic alteration. Consequently, a comprehensive understanding of DNA repair mechanisms will be important for the design of novel interventions for cancer prevention and treatment. This research project aims at defining the fundamental reaction of base- excision repair in higher eukaryotes. Base-excision repair is a major pathway in most living organisms to correct various types of modified bases. Many modified bases can be cytotoxic and mutagenic unless they are repaired. The base-excision repair consists of a sequential process of multistep reactions. The study of this repair reaction will be facilitated by development of an in vitro system. In an initial part of this project, Xenopus laevis oocytes will be used as a model system. The extracts prepared from them have shown high activity in repairing apurinic/apyrimidinic sites which are one of major lesions generated by several damaging agents and are also common intermediate products in base-excision repair. The repair factors participating in this reaction will be isolated by fractionation of these extracts and used to develop a reconstituted system. Factors essential for repair and their interactions during the repair reaction will be characterized in this reconstituted system. Subsequently the results obtained from the study with the Xenopus oocytes will be applied to investigation of the base- excision repair in mammalian systems. Mammalian homologs to the Xenopus repair factors will be obtained on the basis of functional similarity or homology of their amino acid sequences. Characterization of these factors will be performed with human or rodent cultured cells. The results of this study will contribute to a number of aspects in cancer biology such as the mechanism of carcinogenesis and generation of tumor cells resistant to drugs and radiation used for cancer therapy.
