Identification, characterization, and purification of DNA glycosylase/AP lyases by reductive crosslinking to 2 '-deoxyvibooligonucleotides containingspecific base lesions

This paper describes a reductive amination crosslinking protocol that facil itates identification and characterization of a class of DNA repair enzymes , DNA glycosylase/AP lyases, which are involved in base excision repair. Th is crosslinking technique has been used to identify enzymes in crude extrac ts and in partially purified enzyme preparations, to isolate proteins for s equencing, and to confirm the reaction mechanism of members of this enzyme family. Chemical reduction of the Schiff's base enzyme-substrate intermedia te to a stable amine results in the formation of an irreversible covalent b ond between the substrate lesion situated within a 2'-deoxyoligonucleotide and the repair enzyme. This complex can be detected by gel electrophoresis and can also be isolated and analyzed by amino acid sequencing (C) 2000 Aca demic Press.