title: In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells.
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.
authors.affiliation: Institute of Medical Technology and Tampere University Hospital, University of Tampere, 33101 Tampere, Finland.
authors.forename: J N
authors.initials: JN
authors.lastname: Spelbrink
authors.forename: J M
authors.initials: JM
authors.lastname: Toivonen
authors.forename: G A
authors.initials: GA
authors.lastname: Hakkaart
authors.forename: J M
authors.initials: JM
authors.lastname: Kurkela
authors.forename: H M
authors.initials: HM
authors.lastname: Cooper
authors.forename: S K
authors.initials: SK
authors.lastname: Lehtinen
authors.forename: N
authors.initials: N
authors.lastname: Lecrenier
authors.forename: J W
authors.initials: JW
authors.lastname: Back
authors.forename: D
authors.initials: D
authors.lastname: Speijer
authors.forename: F
authors.initials: F
authors.lastname: Foury
authors.forename: H T
authors.initials: HT
authors.lastname: Jacobs
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013367:Submitochondrial Particles / Q000201:enzymology; D014162:Transfection; D018911:Trinucleotide Repeats
chemical_list: D004272:DNA, Mitochondrial; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human