Conditional Targeting of the DNA Repair Enzyme hOGG1 into Mitochondria

Lyudmila I Rachek,Valentina I Grishko,Sergiy I Musiyenko,Mark R Kelley,Susan P Ledoux,Glenn L Wilson

doi:10.1074/jbc.m208770200

Lyudmila I Rachek, Valentina I Grishko + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m208770200

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Abstract

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.

Highlights

The importance of mitochondrial DNA1 for cellular integrity and function is underscored by the fact that a variety of diseases have been associated with mutations in mtDNA, including diabetes [1, 2], ischemic heart disease [3], Parkinson’s disease (4 – 6), Alzheimer’s disease [7,8,9], and the normal process of aging (10 –12)
OGG1 activity decreased slightly over the same time interval. This differential change with age in the mitochondrial and nuclear 8-OxoG glycosylase activities suggests that the expression of these two isoforms may be differentially regulated. These findings indicate that base excision repair (BER) initiated by OGG1 is an essential enzyme required for protection of mtDNA against oxidative damage
Generation of Double-transfected Cell Lines—To fully evaluate the potential of using recombinant hOGG1 to alter mtDNA repair, we used HeLa cells transfected with MTS-OGG1 under the control of a Tet-mediated promoter

Summary

Introduction

The importance of mitochondrial DNA (mtDNA)1 for cellular integrity and function is underscored by the fact that a variety of diseases have been associated with mutations in mtDNA, including diabetes [1, 2], ischemic heart disease [3], Parkinson’s disease (4 – 6), Alzheimer’s disease [7,8,9], and the normal process of aging (10 –12). Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional.

Results

Conclusion