TbKAP6, a Mitochondrial HMG Box-Containing Protein in Trypanosoma brucei, Is the First Trypanosomatid Kinetoplast-Associated Protein Essential for Kinetoplast DNA Replication and Maintenance

Abstract

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei, proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata, some small basic histone H1-like kinetoplast-associated proteins (CfKAP) have been shown to condense isolated kDNA networks in vitro. High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro. These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.T rypanosoma brucei is a unicellular eukaryotic parasite that causes human African trypanosomiasis (HAT) and nagana in livestock in sub-Saharan Africa. The high toxicity of most current chemotherapies and the emergence of drug-resistant parasites are stimulating efforts to identify promising molecular targets and develop next-generation therapeutics (1, 2). These efforts require better understanding of trypanosome basic biology, which differs markedly from that of its host. For instance, trypanosome mitochondrial DNA is organized as a massive chain mail-like network, known as kinetoplast DNA (kDNA). kDNA consists of several thousand minicircles (1 kb) catenated with a few dozen maxicircles (23 kb). This giant planar network is condensed into a disk within the mitochondrial matrix and connected to the flagellar basal body by a transmembrane filament system named the tripartite attachment complex (see the review in reference 3). The lack of a similar DNA network in mammalian cells suggests that kDNA and proteins involved in its metabolism could be appealing therapeutic targets. Indeed, kDNA replication is the primary therapeutic target for ethidium bromide, a drug still used to treat nagana in livestock (4).Trypanosome kDNA replication is unusual in comparison wi...

A dually located multi‐HMG‐box protein of Aspergillus nidulans has a crucial role in conidial and ascospore germination

“…In the tripanosomids Crithidia fasciculata and Trypanosoma cruzi , histone H1‐like proteins are associated with the kinetoplast DNA (Xu et al ., ; de Souza et al ., ), while a proteomic study of the Trypanosma brucei kinetoplast failed to identify any HMG proteins, but revealed, as associated with DNA a protein with no homologues outside trypanosomes (Beck et al ., ). However and in contradiction with the above, a very recent article, describes TbKAP6, an HMG‐box protein of T. brucei associated with the kinetoplast and essential for viability (Wang et al ., ). The process of ‘evolutionary tinkering’ by which proteins of different evolutionary origins are recruited into mitochondrial nucleoids has been discussed by Kucej and Butow ().…”

Characterization of two novel proteins involved in mitochondrial DNA anchoring

“…The repair polymerase PolIA, which is mostly localized in the mitochondrial matrix was not detected. Surprisingly we also found several proteins that have been described to be localized inside the kDNA disc including the gap repair polymerase POL beta PAK and the HMG-box containing proteins KAP3, KAP4 and KAP6 that are thought to fulfill the role of histone proteins in the kDNA (Wang et al, 2014;Xu et al, 1996). Also the third compartment, the antipodal sites, seem to be linked to the TAC even without the kDNA being present.…”

“…Most prominently the kDNA associated proteins KAP3, KAP4 and KAP6. KAP3 was described as a histone H1 like protein in Crithidia fasciculata, while KAP6 is a HMG box containing protein described in T. brucei (Wang et al, 2014;Xu et al, 1996). Bloodstream form trypanosomes have a metabolically much reduced mitochondrion and mutants can be selected that are able to live without a mitochondrial genome.…”

Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA

“…Recent studies showed that certain DNA minor groove binding compounds were able to enter the mitochondrion of Trypanosoma brucei, bind to AT sequences of kDNA and, as a result, cause cell death (60). It was suggested that these minor groove binding compounds may disrupt the functions of kDNA binding proteins, such as the TbKAP6 protein that is essential for kDNA replication and maintenance and also for cell viability (61). TbKAP6 contains two HMG boxes and binds to DNA minor groove (61).…”

“…It was suggested that these minor groove binding compounds may disrupt the functions of kDNA binding proteins, such as the TbKAP6 protein that is essential for kDNA replication and maintenance and also for cell viability (61). TbKAP6 contains two HMG boxes and binds to DNA minor groove (61).…”