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Eukaryotic Cell, April 2005, p. 765-774, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.765-774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Mitochondrial DNA Ligases of Trypanosoma brucei

Nick Downey,{dagger} Jane C. Hines, Krishna M. Sinha, and Dan S. Ray*

Molecular Biology Institute and Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California

Received 23 December 2004/ Accepted 26 January 2005

The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k{alpha} and LIG kß) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k{alpha} and LIG kß are mitochondrial DNA ligases. Epitope-tagged LIG k{alpha} localizes throughout the kDNA, whereas LIG kß shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k{alpha} mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.

* Corresponding author. Mailing address: Paul D. Boyer Hall, 611 Charles Young Dr., University of California, Los Angeles, CA 90095-1570. Phone: (310) 825-4178. Fax: (310) 206-7286. E-mail: danray{at}ucla.edu.

{dagger} Present address: Department of Biology, Luther College, Decorah, Iowa.

Eukaryotic Cell, April 2005, p. 765-774, Vol. 4, No. 4
1535-9778/05/$08.00+0     doi:10.1128/EC.4.4.765-774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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