PMID:15199172
| Citation |
Rachek, LI, Grishko, VI, Alexeyev, MF, Pastukh, VV, LeDoux, SP and Wilson, GL (2004) Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress. Nucleic Acids Res. 32:3240-7 |
|---|---|
| Abstract |
Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases. In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII). To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element. Successful integrations of MTS-EndoIII or MTS-EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot. Western blots of mitochondrial extracts from MTS-EndoIII and MTS-EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent. Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS-EndoIII and MTS-EndoVIII are functional, and both MTS-EndoIII and MTS-EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA. This enhanced repair led to increased cellular resistance to oxidative stress. |
| Links |
PubMed PMC434452 Online version:10.1093/nar/gkh648 |
| Keywords |
Cell Survival; DNA Repair; DNA, Mitochondrial/genetics; Deoxyribonuclease (Pyrimidine Dimer)/genetics; Deoxyribonuclease (Pyrimidine Dimer)/metabolism; Doxycycline/pharmacology; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation; Gene Targeting; HeLa Cells; Humans; Mitochondria/genetics; Mitochondria/metabolism; Oxidative Stress; Recombinant Proteins/metabolism; Transfection |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.