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Published online 15 June 2004

Nucleic Acids Research, 2004, Vol. 32, No. 10 3240-3247
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved

Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress

Lyudmila I. Rachek, Valentina I. Grishko1, Mikhail F. Alexeyev1, Viktoriya V. Pastukh1, Susan P. LeDoux and Glenn L. Wilson*

Department of Cell Biology and Neuroscience and 1 Department of Pharmacology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA

* To whom correspondence should be addressed. Tel: +1 251 460 6765; Fax: +1 251 414 8241; Email: gwilson{at}usouthal.edu

Received April 8, 2004; Revised and Accepted May 20, 2004

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.


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