Akinetoplast is a network of circularDNA (called kDNA) inside amitochondrion that contains many copies of themitochondrial genome.[1][2] The most common kinetoplast structure is a disk, but they have been observed in other arrangements. Kinetoplasts are only found inExcavata of the classKinetoplastida. The variation in the structures of kinetoplasts may reflect phylogenic relationships between kinetoplastids.[3] A kinetoplast is usually adjacent to the organism'sflagellarbasal body, suggesting that it is bound to some components of thecytoskeleton. InTrypanosoma brucei this cytoskeletal connection is called the tripartite attachment complex and includes the proteinp166.[4]
Intrypanosomes, a group of flagellated protozoans, the kinetoplast exists as a dense granule of DNA within the mitochondrion.Trypanosoma brucei, the parasite which causesAfrican trypanosomiasis (African sleeping sickness), is an example of a trypanosome with a kinetoplast. Its kinetoplast is easily visible in samples stained withDAPI, afluorescent DNAstain, or by the use offluorescent in situ hybridization (FISH) with BrdU, athymidine analogue.[5] Another parasite in the genus,Trypanosoma cruzi, causesChagas disease in humans (primarily in Central and South America), which is transmitted through thekissing bug. Although African sleeping sickness is more dangerous than Chagas disease, the kinetoplast ofT. cruzi is significantly larger than that ofT. brucei.[6]Trypanosoma equiperdum causes the diseasedourine inhorses, and is the only sexually transmitted trypanosome infection.[7] The kinetoplasts ofT. equiperdum are unique in that every minicircle has the same genetic sequence.[8]
Purified kinetoplast DNA fromCrithidia fasciculata is sold by two biochemical companies,TopoGen andInspiralis. The kinetoplast DNA is used as a substrate to test the functionality of drugs or toxins targettingTopoisomerase II, a protein associated with cell division that can untangle DNA by passing strands through each other. Since kinetoplasts are normally too large to move throughagar gel, the appearance of bands associated with minicircles during agel electrophoresis assay indicate that Topoisomerase II has decatenated the kinetoplasts. This assay can be used to determine whether drugs or toxins that target Topoisomerase II are present.[9] Kinetoplasts fromC. fasciculata are also used inbiophysical studies of kinetoplast DNA as a natural example of anOlympic gel.[10]
The kinetoplast contains circular DNA in two forms, maxicircles andminicircles. Maxicircles are between 20 and 40kb in size and there are a few dozen per kinetoplast. There are several thousand minicircles per kinetoplast and they are between 0.5 and 1kb in size. Maxicircles encode the typical protein products needed for the mitochondria which is encrypted. Herein lies the only known function of the minicircles - producingguide RNA (gRNA) to decode this encrypted maxicircle information, typically through the insertion or deletion ofuridine residues. The network of maxicircles and minicircles arecatenated to form a planar network that resembleschain mail. Reproduction of this network then requires that these rings be disconnected from the parental kinetoplast and subsequently reconnected in the daughter kinetoplast.[5][11] This unique mode of DNA replication may inspire potentialdrug targets.
The best studied kDNA structure is that ofCrithidia fasciculata, a catenated disk of circular kDNA maxicircles and minicircles, most of which are notsupercoiled.[3] Exterior to the kDNA disk but directly adjacent are two complexes of proteins situated 180˚ from each other and are involved in minicircle replication.[1][2][5][11] The networktopology of kinetoplast DNA is primarily understood from experiments onC. fasciculata,[12] based on gel electrophersis of kinetoplasts that have been broken down byrestriction enzymes. These experiments indicate that each minicircle is linked to three on average, and that thecrystal lattice structure most consistent with the data is thehoneycomb lattice. More recent studies based onatomic force microscopy have corroborated the trivalent connectivity, but have shown that the structure is highly disordered.[13]
Variations of kinetoplast networks have also been observed and are described by the arrangement and location of their kDNA.
The presence of this variety of kDNA structures reinforces the evolutionary relationship between the species of kinetoplastids. As pan-kDNA most closely resembles a DNAplasmid, it may be the ancestral form of kDNA.[3]
The replication of the kinetoplast occurs simultaneously to the duplication of the adjacent flagellum and just prior to thenuclear DNA replication. In a traditionalCrithidia fasciculata kDNA network, initiation of replication is promoted by the unlinking of kDNA minicircles viatopoisomerase II. The free minicircles are released into a region between the kinetoplast and the mitochondrial membrane called the kinetoflagellar zone (KFZ).[2][3][11] After replication the minicircles migrate by unknown mechanisms to the antipodal protein complexes that contain several replication proteins including anendonuclease,helicase,DNA polymerase,DNA primase, andDNA ligase, which initiate repair of remaining discontinuities in the newly replicated minicircles.[5]
This process occurs one minicircle at a time, and only a small number of minicircles are unlinked at any given moment. To keep track of which minicircles have been replicated, upon rejoining to the kDNA network a small gap remains in the nascent minicircles, which identifies them as having already been replicated. Minicircles that have not yet been replicated are still covalently closed. Immediately after replication, each progeny is attached to the kDNA network proximal to the antipodal protein complexes and the gaps are partially repaired.[1][11]
As minicircle replication progresses, to prevent the build-up of new minicircles, the entire kDNA network will rotate around the central axis of the disk. The rotation is believed to be directly connected to the replication of the adjacent flagellum, as the daughter basal body will also rotate around the mother basal body in a timing and manner similar to the rotation of the kinetoplast. By rotating, the minicircles of the daughter kinetoplast are assembled in a spiral fashion and begin moving inward toward the center of the disk as new minicircles are unlinked and moved into the KFZ for replication.[2][5][11]
While the exact mechanisms for maxicircle kDNA have yet to be determined in the same detail as minicircle kDNA, a structure called anabelschnur (German for "umbilical cord") is observed that tethers the daughter kDNA networks but eventually breaks during separation. Using FISH probes to target the nabelschnur, it has been found to contain maxicircle kDNA.[5]
Kinetoplast replication is described as occurring in five stages, each in relation to the replication of the adjacent flagellum.
Trypanosoma cruzi is able to repair nucleotides in its genomic or kinetoplastDNA that have been damaged byreactive oxygen species produced by the parasite's host during infection.[14]DNA polymerase beta expressed inT. cruzi is employed in the removal of oxidativeDNA damages by the process ofbase excision repair. It appears that DNA polymerase beta acts during kinetoplastDNA replication to repair oxidative DNA damages induced bygenotoxic stress in this organelle.[14]