Identification, Characterization, and Purification of DNA Glycosylase/AP Lyases by Reductive Crosslinking to 2-Deoxyribooligonucleotides Containing Specific Base Lesions

This paper describes a reductive amination crosslinking protocol that facilitates identification and characterization of a class of DNA repair enzymes, DNA glycosylase/AP lyases, which are involved in base excision repair. This crosslinking technique has been used to identify enzymes in crude extracts and in partially purified enzyme preparations, to isolate proteins for sequencing, and to confirm the reaction mechanism of members of this enzyme family. Chemical reduction of the Schiffʹs base enzyme–substrate intermediate to a stable amine results in the formation of an irreversible covalent bond between the substrate lesion situated within a 2ʹ-deoxyoligonucleotide and the repair enzyme. This complex can be detected by gel electrophoresis and can also be isolated and analyzed by amino acid sequencing.