IDENTIFICATION OF 5'-DEOXYRIBOSE PHOSPHATE LYASE ACTIVITY IN HUMAN DNA-POLYMERASE-GAMMA AND ITS ROLE IN MITOCHONDRIAL BASE EXCISION-REPAIR IN-VITRO

Mitochondria have been proposed to possess base excision repair proces ses to correct oxidative damage to the mitochondrial genome. As the on ly. DNA polymerase (pol) present in mitochondria, pol gamma is necessa rily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-pro voked base excision repair reconstituted in vitro with purified compon ents. Subsequent to actions of uracil-DNA glycosylase and apurinic/apy rimidinic endonuclease, human pol gamma was able to fill a single nucl eotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP ) flap. We report here that the catalytic subunit of human pol gamma c atalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensi onal protein structure. The dRP lyase activity does not require divale nt metal ions, and the ability to trap covalent enzyme-DNA complexes w ith NaBH4 strongly implicates a Schiff base intermediate in a beta-eli mination reaction mechanism.