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Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

Joachim Kienhöfer^*, Dagmar Johanna Franziska Häussler, Florian Ruckelshausen^*, Elisabeth Muessig^*, Klaus Weber, David Pimentel, Volker Ullrich^*, Alexander Bürkle^* and Markus Michael Bachschmid^,1

^* Department of Biology, University of Konstanz, Konstanz, Germany;

Vascular Biology Unit and

Myocardial Unit, Whitaker Cardiovascular Institute, Boston, Massachusetts, USA; and

Herz-Zentrum Bodensee, Kreuzlingen, Switzerland

¹ Correspondence: Boston University Medical Center, Vascular Biology Unit X726, 650 Albany St., Boston, MA 02118, USA. E-mail: bach{at}bu.edu

Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2–8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase (Pol), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Pol were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.—Kienhöfer, J., Häussler, D. J. F., Ruckelshausen, F., Muessig, E., Weber, K., Pimentel, D., Ullrich, V., Bürkle, A., Bachschmid, M. M. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents.

Key Words: SOD2 • nucleoid complex • aging • smooth muscle cell • TFAM • Pol

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