Base Excision Repair Assay Using Xenopus laevis Oocyte Extracts

Base excision repair is a major mechanism for correcting modified bases. The first step of this repair mechanism is the removal of a modified base by a specific DNA-N -glycosylase to leave an apurinic/apyrimidinic (AP) site. Subsequently, the AP site is repaired through sequential reactions, including incision of the DNA backbone, excision of the deoxyribose phosphate (dRP), and DNA synthesis and ligation. Since we found that an extract from Xenopus laevis oocytes efficiently repaired AP sites (1 ), this repair mechanism has been further analyzed in detail. As a result, it turns out that base excision repair is carried out by two distinct pathways, the proliferating cell nuclear antigen-(PCNA) dependent pathway and the DNA polymerase β- (pol β) dependent pathway (2 ). Subsequent studies from several laboratories revealed that the two alternative pathways for base excision repair are observed not only in X. laevis oocyte extracts, but also in mammalian cell extracts (3 ,4 ; see Chapter 24 ). Thus, the in vitro system with X. laevis oocyte extracts can serve as a model system for base excision repair in vertebrates.