In Vivo Functional Analysis of the Human Mitochondrial DNA Polymerase POLG Expressed in Cultured Human Cells

Johannes N Spelbrink,Janne M Toivonen,Gerrit A.J Hakkaart,Johanna M Kurkela,Helen M Cooper,Sanna K Lehtinen,Nicolas Lecrenier,Jaap Willem Back,Dave Speijer,Françoise Foury,Howard T Jacobs

doi:10.1074/jbc.m000559200

Abstract

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.

Highlights

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented
By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase
Tagged POLG Is Targeted to Mitochondria in Vivo—To confirm that the POLG protein is targeted to mitochondria we constructed reporter gene fusions of full-length POLG, fused at the COOH terminus to a Myc tag (POLG-myc), or to green fluorescent protein (POLG-GFP)

Summary

Introduction

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities. The human POLG gene encodes the catalytic subunit of what is believed to be the only DNA polymerase active in mitochondria, polymerase ␥, POLG (4 – 6). DNA replication requires the combined action of many proteins, including those involved in nucleotide metabolism, DNA unwinding, priming of DNA synthesis, decatenation, and sta-

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