HUMAN MITOCHONDRIAL URACIL-DNA GLYCOSYLASE PREFORM (UNG1) IS PROCESSED TO 2 FORMS ONE OF WHICH IS RESISTANT TO INHIBITION BY AP SITES

The preform of human mitochondrial uracil-DNA glycosylase (UNG1) conta ins 35 N-terminal residues required for mitochondrial targeting. We ha ve examined processing of human UNG1 expressed in insect cells and pro cessing in vitro by human mitochondrial extracts, In insect cells we d etected a major processed form lacking 29 of the 35 unique N-terminal residues (UNG1 Delta 29, 31 kDa) and two minor forms lacking the 75 an d 77 N-terminal residues, respectively (UNG1 Delta 75 and UNG1 Delta 7 7, 26 kDa), Purified UNG1 Delta 29 was effectively cleaved in vitro to a fully active 26 kDa form by human mitochondrial extracts. Furthermo re, endogenous forms of 31 and 26 kDa were also observed in HeLa mitoc hondrial extracts. The sequences at the cleavage sites, as identified by peptide sequencing, were compatible with the known specificity of m itochondrial processing peptidase (MPP), However, in vitro cleavage of UNG1 Delta 29 by mitochondrial extracts did not require divalent cati ons and was stimulated by EDTA, indicating the involvement of a proces sing peptidase distinct from MPP at the second site. Interestingly, wh ile UNG1 Delta 29 generally has the typical properties reported for ot her uracil-DNA glycosylases, it is not inhibited by apurinic/apyrimidi nic sites. Our results indicate that the preform of human mitochondria l uracil-DNA glycosylase is processed to distinctly different forms la cking 29 or 75/77 N-terminal residues, respectively.