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

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Spelbrink, Johannes Toivonen, Janne Hakkaart, Gerrit Kurkela, Johanna Cooper, Helen Lecrenier, Nicolas [UCL] Foury, Françoise [UCL] Jacobs, Howard

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in Dice 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.

Document type Article de périodique (Journal article) – Article de recherche
Access type Accès restreint
Publication date 2000
Language Anglais
Journal information "Journal of Biological Chemistry" - Vol. 275, no. 32, p. 24818-24828 (2000)
Peer reviewed yes
Publisher Amer Soc Biochemistry Molecular Biology Inc (Bethesda)
issn 0021-9258
e-issn 1083-351X
Publication status Publié
Affiliation UCL - MD/BICL - Département de biochimie et de biologie cellulaire
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Bibliographic reference Spelbrink, Johannes ; Toivonen, Janne ; Hakkaart, Gerrit ; Kurkela, Johanna ; Cooper, Helen ; et. al. In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. In: Journal of Biological Chemistry, Vol. 275, no. 32, p. 24818-24828 (2000)
Permanent URL http://hdl.handle.net/2078.1/43383