Minicircular kinetoplast DNA from Trypanosomatidae

Analysis of primary structure and organization of mitochondrial (kinetoplast) DNA of flagellates occupies a prominent place in the studies of eukaryote mitochondrial genomes, owing to its unusual organization and functioning as well as to the epidemiological role of the Trypanosomatidae family. According to contemporary notions, living zooflagellates are direct descendants of the ancestral forms that gave rise to all eukaryotic kingdoms. Hence, comparative mtDNA studies of recent Trypanosomatidae open broad prospects for phylogenetic reconstructions and analysis of presumable routes of eukaryote evolution. The structure, characteristics, and functions of Trypanosomatidae minicircular kinetoplast DNA are discussed here.     

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.     

Characteristics of kinetoplastic DNA from Leptomonas pessoai

The kinetoplast (mitochondrial) DNA from trypanosomatid Leptomonas pessoai represents a network, composed of mini-circles heterogeneous in base sequence and homogeneous maxi-circles and thus has the main structural features in common with DNAs from kinetoplasts of other Trypanosomatidae. The size of mini-circular molecules of DNA is 1,35 kilobase pairs (kbp) and that of maxicircular molecules-30,9 kbp. Based on the data of single and double restriction cleavages the physical map of the maxi-circular molecules was constructed for the endonucleases BamHI, BglII, BspI , HindIII, MspI, SalGI and PstI.     

The Differentiation of Herpetomonas megaseliae: Ultrastructural Observations*

Ultrastructure of both undifferentiated (promastigote and paramastigote) and differentiated (opisthomastigote) forms of Herpetomonas megaseliae is described. There is a posterior migration of the kinetoplast at the end of the exponential growth phase. The posterior extension of the flagellar pocket precedes migration of the kinetoplast. Opisthomastigotes have an electron-translucent mitochondrial matrix in comparison with undifferentiated forms. The Golgi body changes from a stack of flattened sacs to an aggregation of vesicles. Several structures previously reported from Trypanosomatidae, e.g. subpellicular organelles, pellicular microtubules, membrane whorls, stored metabolic products, surface blebs, and an intraflagellar body are also present in H. megaseliae.     

THE LOSS OF KINETOPLASTIC DNA IN TWO SPECIES OF TRYPANOSOMATIDAE TREATED WITH ACRIFLAVINE

The effects of acriflavine on two species of Trypanosomatidae, Crithidia luciliae and Trypanosoma mega, have been investigated. It has been observed that kinetoplastic (i.e. mitochondrial) DNA is lost in a high percentage of acriflavine-treated cells. Resting flagellates, from stationary-phase or hemin-deficient cultures, are considerably more resistant to the acridine than are flagellates from a log-phase culture. When the kinetoplast has retained some DNA and still remains visible in stained smears, it appears reduced in size, and its ultrastructure is extremely abnormal: the DNA fibrils, clearly visible in normal kinetoplasts, are condensed; they appear as an electron-opaque, apparently homogeneous mass, separated from the membranes by a space of low electron-opacity. Analyses of DNA extracts, with high speed centrifugation in CsCl density gradients, revealed that the satellite band, presumably kinetoplastic DNA, is lost by trypanosomes grown for 5 days in the presence of acriflavine. Radioautography was used to study the effects of acriflavine on thymidine-³H incorporation in C. luciliae. At the concentration which affects the kinetoplast specifically, the dye produces an 87% inhibition of thymidine incorporation in this organelle. The kinetics of this inhibition suggest a direct effect on replication. No decrease in incorporation occurs in the nucleus. These results lead to the conclusion that loss of kinetoplastic DNA is due to continued growth and cell division in the absence of kinetoplastic DNA replication. Several hypotheses are discussed concerning the specificity of the dye's action upon the replication of extrachromosomal DNA.     

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.     

Role of p38 in replication of Trypanosoma brucei kinetoplast DNA

Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as theta-structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.     

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.     

Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication

ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.     

Isolation and characterization of circular DNA molecules heterogeneous in size from a dyskinetoplastic strain of Trypanosoma equiperdum

In a naturally occuring dyskinetoplastic mutant strain of $\text{T. equiperdum}$, covalently closed circular DNA molecules of assumed mitochondrial origin were isolated. These molecules, heterogeneous in size, represent 6–9 % of total DNA and are essentially organized in catenated oligomers composed of molecules of different length. The typical molecular organization of the kinetoplast DNA from kinetoplastic trypanosomes, the network, was not observed.     

A high-order trans-membrane structural linkage is responsible for mitochondrial genome positioning and segregation by flagellar basal bodies in trypanosomes

In trypanosomes, the large mitochondrial genome within the kinetoplast is physically connected to the flagellar basal bodies and is segregated by them during cell growth. The structural linkage enabling these phenomena is unknown. We have developed novel extraction/fixation protocols to characterize the links involved in kinetoplast-flagellum attachment and segregation. We show that three specific components comprise a structure that we have termed the tripartite attachment complex (TAC). The TAC involves a set of filaments linking the basal bodies to a zone of differentiated outer and inner mitochondrial membranes and a further set of intramitochondrial filaments linking the inner face of the differentiated membrane zone to the kinetoplast. The TAC and flagellum-kinetoplast DNA connections are sustained throughout the cell cycle and are replicated and remodeled during the periodic kinetoplast DNA S phase. This understanding of the high-order trans-membrane linkage provides an explanation for the spatial position of the trypanosome mitochondrial genome and its mechanism of segregation. Moreover, the architecture of the TAC suggests that it may also function in providing a structural and vectorial role during replication of this catenated mass of mitochondrial DNA. We suggest that this complex may represent an extreme form of a more generally occurring mitochondrion/cytoskeleton interaction.     

The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast

The kinetoplast is a concatenated network of circular DNA molecules found in the mitochondrion of many trypanosomes. This mass of DNA is replicated in a discrete "S" phase in the cell cycle. We have tracked the incorporation of the thymidine analogue 5-bromodeoxyuridine into newly replicated DNA by immunofluorescence and novel immunogold labeling procedures. This has allowed the detection of particular sites of replicated DNA in the replicating and segregating kinetoplast. These studies provide a new method for observing kinetoplast DNA (kDNA) replication patterns at high resolution. The techniques reveal that initially the pattern of replicated DNA is antipodal and can be detected both on isolated complexes and in replicating kDNA in vivo. In Trypanosoma brucei the opposing edges of replicating kDNA never extend around the complete circumference of the network, as seen in other kinetoplastids. Furthermore, crescent-shaped labeling patterns are formed which give way to labeling of most of the replicating kDNA except the characteristic midzone. The configuration of these sites of replicated DNA molecules is different to previous studies on organisms such as Crithidia fasciculata, suggesting differences in the timing of replication of mini and maxicircles and/or organization of the replicative apparatus in the kinetoplast of the African trypanosome.     

A new function of Trypanosoma brucei mitochondrial topoisomerase II is to maintain kinetoplast DNA network topology

The mitochondrial genome of Trypanosoma brucei, called kinetoplast DNA, is a network of topologically interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles and reattachment of the progeny. Here we report a new function of the mitochondrial topoisomerase II (TbTOP2mt). Although traditionally thought to reattach minicircle progeny to the network, here we show that it also mends holes in the network created by minicircle release. Network holes are not observed in wild‐type cells, implying that this mending reaction is normally efficient. However, RNAi of TbTOP2mt causes holes to persist and enlarge, leading to network fragmentation. Remarkably, these network fragments remain associated within the mitochondrion, and many appear to be appropriately packed at the local level, even as the overall kinetoplast organization is dramatically altered. The deficiency in mending holes is temporally the earliest observable defect in the complex TbTOP2mt RNAi phenotype.     

Antiparasitic compounds that target DNA

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.     

Large circular mitochondrial DNA in Crithidia luciliae

A novel circular DNA, 11.3 μm in contour length, has been found in a pure kinetoplast DNA fraction of Crithidia luciliae. The mitochondrial nature of the kinetoplast and the absence of these large circular molecules in the nuclear fraction of DNA suggest that they constitute the mitochondrial genome of this species.