A nonisotopic assay for unambiguous assignment of DNA glycosylase functionality.

DNA glycosylase assays still commonly involve the use of radioactively end-labeled DNA substrates, although in recent years a few nonradioactive methods have been described [1–3]. All reported assays, however, have in common that they are strand cleavage assays. In contrast to DNA glycosylases with inherent strand-scission activity (i.e., DNA glycosylases/AP-lyases), monofunctional DNA glycosylases do not cleave the DNA backbone but leave behind an abasic or apurinic/apyrimidinic (AP) site in DNA [4]. Thus, for the analysis of monofunctional DNA glycosylase products with those assays, a posttreatment of samples is necessary to induce strand scission adjacent to the abasic site. The lack of current DNA glycosylase assays to concurrently detect abasic DNA products and AP-lyase products has likely been a contributing factor in controversies on the classification of some DNA glycosylases such as the adenine glycosylase MutY [5–7]. Indeed, it has been suggested that conflicting glycosylase assay results may have been caused by artifacts arising from different handling of DNA samples (heat or base treatment) and/or high temperature of gels during denaturing PAGE, taking into account the inherent lability of abasic sites [6,7]. Another limitation of current DNA glycosylase assays is that DNA substrates are restricted to relatively short DNA duplexes (typically 630 bp). The use of longer DNA substrates may allow, for instance, investigations aimed at delineating the mecha-