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

• Published in: Current biology 1999-02, Vol.9 (4), p.174-185
• Main Authors: Haushalter, Karl A., Todd Stukenberg, P., Kirschner, Marc W., Verdine, Gregory L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cloning, Molecular; Conserved Sequence; DNA Glycosylases; Humans; Molecular Sequence Data; N-Glycosyl Hydrolases - chemistry; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; Protein Conformation; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Uracil-DNA Glycosidase; Xenopus; Xenopus Proteins
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/S0960-9822(99)80087-6

Background: The cellular environment exposes DNA to a wide variety of endogenous and exogenous reactive species that can damage DNA, thereby leading to genetic mutations. DNA glycosylases protect the integrity of the genome by 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 expressed DNA glycosylases, based on their ability to bind to a library of four synthetic inhibitors that target the enzyme's active site. These inhibitors, used in conjunction with the in vitro expression cloning procedure, led to the identification of novel Xenopus and human proteins, xSMUG1 and hSMUG1, respectively, that efficiently excise uracil residues from DNA. Despite a lack of statistically significant overall sequence similarity to the two established classes of uracil-DNA glycosylases, the SMUG1 enzymes contain motifs that are hallmarks of a shared active-site structure and overall protein architecture. The unusual preference of SMUG1 for single-stranded rather than double-stranded DNA suggests a unique biological function in ridding the genome of uracil residues, which are potent endogenous mutagens. Conclusions: The ‘proteomics’ approach described here has led to the isolation of a new family of uracil-DNA glycosylases. The three classes of uracil-excising enzymes (SMUG1 being the most recently discovered) represent a striking example of structural and functional conservation in the almost complete absence of sequence conservation.