Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil-DNA glycosylase.

Gene-targeted mice deficient in the evolutionarily conserved uracil-DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung(-) mutants. A complementary uracil-DNA glycosylase activity detected in ung(-/-) murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil-DNA glycosylase in UNG-deficient mice. SMUG1 is present at similar levels in cell nuclei of non-proliferating and proliferating tissues, indicating a replication- independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti-mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.

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 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.