In vivo functional analysis of the human mitochondrial DNA polymerase POLGexpressed in cultured human cells

The human gene POLG encodes the catalytic subunit of mitochondrial DNA poly merase, but its precise roles in mtDNA metabolism in Dice have not hitherto been documented. By expressing POLG fusion proteins in cultured human cell s, we show that the enzyme is targeted to mitochondria, where the Myc epito pe-tagged POLG is catalytically active as a DNA polymerase. Long-term cultu re of cells expressing wild-type POLG-myc revealed no alterations in mitoch ondrial function. Expression of POLG-myc mutants created dominant phenotype s demonstrating important roles for the protein in mtDNA maintenance and in tegrity. The D198A amino acid replacement abolished detectable 3'-5' (proof reading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous cultur e. Further culture resulted in the selection of cells with an inactivated m utator polymerase, and a reduced mutation load in mtDNA Transient expressio n of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial D NA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expr ession. These findings demonstrate that POLG exonuclease and polymerase fun ctions are essential for faithful mtDNA maintenance in vivo, and indicate t he importance of key residues for these activities.