New immunoaffinity-LC-MS/MS methodology reveals that Aag null mice are deficient in their ability to clear 1, N6-etheno-deoxyadenosine DNA lesions from lung and liver in vivo

Abstract

The mouse alkyladenine DNA glycosylase (Aag) initiates base excision repair with a broad substrate range that includes the highly mutagenic exocyclic etheno DNA base adduct 1, N 6-ethenodeoxyadenosine (εdA). Previous attempts to determine the in vivo role of Aag in εdA repair were complicated by technological difficulties in measuring low levels of εdA in genomic DNA. Here we describe the development of a new method for εdA detection in genomic DNA that couples an immunoaffinity purification with LC-MS/MS analysis and that utilizes an isotopically labeled internal standard. We go on to describe the application of this method to measuring the in vivo repair of εdA base lesions in liver and lung tissue of wild type and Aag null mice. Our results demonstrate that while Aag clearly represents the major DNA repair enzyme for the in vivo removal εdA bases, these lesions can also be eliminated from the genome via an alternative mechanism.