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

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. Topoisomerase II (topo II)1 is a ubiquitous enzyme that catalyzes strand passing of double-stranded DNA in an ATP-dependent manner. It is involved in a number of processes, including replication and transcription of nuclear and mitochondrial DNA, chromosome segregation, and chromatin organization (1).Topo II is an important component of the kinetoplast-mitochondrion organelle of the kinetoplastid protozoa. These flagellates represent one of the most primitive groups of eukaryotes, equipped with a number of unique features. Their mitochondrial DNA, termed kinetoplast (k) DNA, contains up to 40% of total cellular DNA and is composed of thousands of minicircles and dozens of maxicircles. Maxicircles encode mitochondrial genes, the transcripts of which undergo extensive editing of the uridine insertion/deletion type. Minicircles bear guide RNA genes, which provide information for the editing process (for recent review see Refs. 2 and 3).The order Kinetoplastida is divided into the suborder Bodonina, which comprises free-living commensalic or parasitic biflagellated species, and the suborder Trypanosomatina, members of which are equipped with a single flagellum and are obligatory parasitic. In the mitochondrion of Trypanosomatina, the DNA molecules are present as relaxed circles catenated into a single giant network (for recent review see Refs. 4 and 5). So far, topo II was studied in detail in the model trypanosomatids Crithidia fasciculata and Trypanosoma brucei, where it is involved in decatenation, replication, and recatenation of the kDNA minicircles (6 -8). The enzyme has also a very characteristic localization in two opposing antipodal protein centers (8). Topoisomerases are considered to be a...

Comparative Metabolism of Free‐living Bodo saltans and Parasitic Trypanosomatids

“…So far, only a few individual enzymes have been described and characterized in B. saltans (Brown et al. ; Gažiová and Lukeš ; Jackson et al. ; Lawrie ), and the RNA editing machinery performing insertions and/or deletions of uridines is apparently present in its mitochondrion (Blom et al.…”

Molecular characterization of type II topoisomerase in the endosymbiont-bearing TrypanosomatidBlastocrithidia culicis

“…By contrast, the antiserum raised against the recombinant T. cruzi located topoII in the nucleus of T. cruzi and C. fasciculata (Fragoso et al , 1998). In Leishmania donovanni and Bodo saltans , polyclonal antibodies raised against their respective topoII enzymes, labeled both the kinetoplast and the nucleus, suggesting that the same enzyme is present in both compartments (Das et al , 2001; Gaziová & Lukes, 2003). We have also shown that DNA gyrase inhibitors cause dramatic changes in the kDNA network, suggesting that topoisomerase II is important for maintaining the kDNA structure (Cavalcanti et al , 2004).…”

“…Topoisomerase activity has been observed in a number of kinetoplastida, including Trypanosoma cruzi (Douc‐Rasy et al , 1986), Leishmania donovani (Chakraborty and Majumder, 1987) and Crithidia fasciculata (Melendy & Ray, 1989). Furthermore, TOP2 genes have been characterized for Trypanosoma brucei (Strauss & Wang, 1990), T. cruzi (Fragoso & Goldenberg, 1992), C. fasciculata (Pasion et al , 1992), L. donovani (Das et al , 2001), L. infantum (Hanke et al , 2003) and Bodo saltans (Gaziová & Lukes, 2003).…”