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Molecular and Cellular Biology, August 2008, p. 4975-4987, Vol. 28, No. 16
0270-7306/08/$08.00+0     doi:10.1128/MCB.00457-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Removal of Oxidative DNA Damage via FEN1-Dependent Long-Patch Base Excision Repair in Human Cell Mitochondria {triangledown} ,{dagger}

Pingfang Liu,1 Limin Qian,2 Jung-Suk Sung,3 Nadja C. de Souza-Pinto,4 Li Zheng,2 Daniel F. Bogenhagen,5 Vilhelm A. Bohr,4 David M. Wilson III,4 Binghui Shen,2* and Bruce Demple1*

Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115,1 Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010,2 Department of Biology, Dongguk University, Seoul 100-715, South Korea,3 Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224,4 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-86515

Received 19 March 2008/ Returned for modification 1 May 2008/ Accepted 29 May 2008

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase {gamma} and interferes with SP-BER by covalently trapping polymerase {gamma} during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

* Corresponding author. Mailing address for Binghui Shen: Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010. Phone: (626) 301-8878. Fax: (626) 301-8280. E-mail: bshen{at}coh.org. Mailing address for Bruce Demple: Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115. Phone: (617) 432-3462. Fax: (617) 432-2590. E-mail: bdemple{at}hsph.harvard.edu

{triangledown} Published ahead of print on 9 June 2008.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, August 2008, p. 4975-4987, Vol. 28, No. 16
0270-7306/08/$08.00+0     doi:10.1128/MCB.00457-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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