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Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800. This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting. To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration. An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene. The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting. Gel filtration gave essentially similar estimates. An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence. Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei. Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei. We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.