Conditional Targeting of the DNA Repair Enzyme hOGG1 into Mitochondria

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 d...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-11, Vol.277 (47), p.44932-44937
Main Authors: Rachek, Lyudmila I, Grishko, Valentina I, Musiyenko, Sergiy I, Kelley, Mark R, LeDoux, Susan P, Wilson, Glenn L
Format: Article
Language:English
Subjects:
Animals
Cell Division
Cell Fractionation
Cell Survival
DNA Repair
DNA, Mitochondrial - genetics
DNA-Formamidopyrimidine Glycosylase
Doxycycline - metabolism
Gene Expression Regulation, Enzymologic
HeLa Cells
Humans
Mitochondria - genetics
Mitochondria - metabolism
N-Glycosyl Hydrolases - genetics
N-Glycosyl Hydrolases - metabolism
Protein Transport - physiology
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Regulatory Sequences, Nucleic Acid
Transfection
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M208770200