Identification of a new uracil-DNA glycosylase family by expression cloning using synthetic inhibitors

Background: The cellular environment exposes DNA to a wide variety of endog enous and exogenous reactive species that can damage DNA, thereby leading t o genetic mutations. DNA glycosylases protect the integrity of the genome b y catalyzing the first step in the base excision-repair of lesions in DNA. Results: Here, we report a strategy to conduct genome-wide screening for ex pressed DNA glycosylases, based on their ability to bind to a library of fo ur synthetic inhibitors that target the enzyme's active site. These inhibit ors, used in conjunction with the in vitro expression cloning procedure, le d to the identification of novel Xenopus and human proteins, xSMUG1 and hSM UG1, respectively, that efficiently excise uracil residues from DNA. Despit e a lack of statistically significant overall sequence similarity to the tw o established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall pr otein architecture. The unusual preference of SMUG1 for single-stranded rat her than double-stranded DNA suggests a unique biological function in riddi ng the genome of uracil residues, which are potent endogenous mutagens. Conclusions: The 'proteomics' approach described here has led to the isolat ion of a new family of uracil-DNA glycosylases, The three classes of uracil -excising enzymes (SMUG1 being the most recently discovered) represent a st riking example of structural and functional conservation in the almost comp lete absence of sequence conservation.