| Abstract
| - The preform of human mitochondrial uracil-DNA glycosylase (UNG1) contains 35 N-terminal residues required for mitochondrial targeting. We have examined processing of human UNG1 expressed in insect cells and processing in vitro by human mitochondrial extracts. In insect cells we detected a major processed form lacking 29 of the 35 unique N-terminal residues (UNG1Δ29, 31 kDa) and two minor forms lacking the 75 and 77 N-terminal residues, respectively (UNG1Δ75 and UNG1Δ77, 26 kDa). Purified UNG1Δ29 was effectively cleaved in vitro to a fully active 26 kDa form by human mitochondrial extracts. Furthermore, endogenous forms of 31 and 26 kDa were also observed in HeLa mitochondrial extracts. The sequences at the cleavage sites, as identified by peptide sequencing, were compatible with the known specificity of mitochondrial processing peptidase (MPP). However, in vitro cleavage of UNG1Δ29 by mitochondrial extracts did not require divalent cations and was stimulated by EDTA, indicating the involvement of a processing peptidase distinct from MPP at the second site. Interestingly, while UNG1Δ29 generally has the typical properties reported for other uracil-DNA glycosylases, it is not inhibited by apurinic/apyrimidinic sites. Our results indicate that the preform of human mitochondrial uracil-DNA glycosylase is processed to distinctly different forms lacking 29 or 75/77 N-terminal residues, respectively.
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