Mitochondrial and Nuclear Localization of Topoisomerase II in the Flagellate Bodo saltans (Kinetoplastida), a Species with Non-catenated Kinetoplast DNA

Ivana Gažiová,Julius Lukeš

doi:10.1074/jbc.m202347200

Ivana Gažiová, Julius Lukeš

Open Access

https://doi.org/10.1074/jbc.m202347200

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Abstract

We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.

Highlights

Topo II is an important component of the kinetoplast-mitochondrion organelle of the kinetoplastid protozoa
Phylogenetic analysis based on the sequence of the entire topoisomerase II (topo II) gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids
Isolation of the Topo II Gene and Sequence Analysis—To isolate the protein-coding region of the kinetoplast-associated type II topoisomerase, we designed degenerate oligonucleotides based on the multiple alignment of available topo II sequences of the trypanosomatids T. brucei, C. fasciculata, and T. cruzi, and PCR-amplified a conserved 1.5-kb-long ATP-binding region located in the N-terminal part of the gene (Fig. 1A)

Summary

Introduction

Topo II is an important component of the kinetoplast-mitochondrion organelle of the kinetoplastid protozoa. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate.

Results

Conclusion