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J. Biol. Chem., Vol. 281, Issue 27, 18499-18506, July 7, 2006

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Overexpression of a Cytochrome b₅ Reductase-like Protein Causes Kinetoplast DNA Loss in Trypanosoma brucei^*

From the Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163–167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b₅ reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b₅ reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b₅ reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.

Received for publication, March 27, 2006

^* This work was supported by National Institutes of Health Grant AI058613. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Molecular Microbiology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.

² To whom correspondence should be addressed: Dept. of Biological Chemistry, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3790; Fax: 410-955-7810; E-mail: penglund{at}jhmi.edu.

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