Unlocking the Secrets of Trypanosome Kinetoplast DNA Network Replication

Klingbeil, Michele M.; Drew, Mark E.; Liu, Yanan; Morris, James C.; Motyka, Shawn A.; Saxowsky, Tina T.; Wang, Zefeng; Englund, Paul T.

doi:10.1078/1434-4610-00066

Cited by 49 publications

(49 citation statements)

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“…Our electron microscopy analysis strongly suggests that the kinetoplast/ basal body connection is maintained via the TAC during all stages of the cell cycle, but it is important to provide corroborative evidence by using different approaches. The situation during S phase is particularly intriguing because kDNA replication is known to involve the release of at least one component (minicircles) from the kinetoplast complex (Abu-Elneel et al, 2001;Drew and Englund, 2001;Klingbeil et al, 2001;Morris et al, 2001). This raises the question of whether the kinetoplast matrix structure as a whole is still firmly attached to the basal bodies during S phase by means of the TAC.…”

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confidence: 99%

“…Our correlation of the known cell cycle markers in T. brucei and the presence of these fibrous lobes place their occurrence within S phase. Replication of the T. brucei kDNA network occurs with maxicircles replicating within the structure, whereas minicircles leave and reattach at the two opposing poles of the network (Ferguson et al, 1994;Robinson and Gull, 1994;Abu-Elneel et al, 2001;Drew and Englund, 2001;Klingbeil et al, 2001;Morris et al, 2001). In addition to replicated minicircles, these antipodal sites contain some of the enzymes necessary for kDNA replication.…”

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confidence: 99%

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A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Ogbadoyi

Robinson

Gull

2003

MBoC

234

279

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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. INTRODUCTIONThe African trypanosome Trypanosoma brucei belongs to a large order of pathogenic and free-living flagellated protozoa designated the kinetoplastida. The majority of these organisms possess a single mitochondrion containing a structural mass of proteins and catenated circular DNA molecules, the kinetoplast. The kinetoplast is essential for survival and cyclical transmission of the parasite between mammalian host and tsetse fly. Early light microscope descriptions of trypanosomes recognized the precise location of the kinetoplast in proximity to the base of the flagellum (Robertson, 1912(Robertson, , 1913. The positions of the flagellum and kinetoplast have become the central features in classifying the different life cycle stages of kinetoplastids (Hoare and Wallace, 1966;Vickerman, 1976). Electron microscopy revealed the kinetoplast to be a highly complex disk-shaped structure located within a distended portion of the mitochondrial matrix adjacent to the flagellum basal bodies (Vickerman, 1973). The T. brucei kinetoplast consists of multiple copies of topologically interlocked circular DNA molecules termed maxicircles and minicircles, along with specific kinetoplast proteins. The maxicircle composition of the T. brucei network is 25-50 copies and represents 10% of the network mass (each maxicircle is 20 kb and all have identical DNA sequence). Maxicircles encode the mitochondrial proteins and some guide RNAs (gRNAs), whereas the minicircles are present in several thousand copies (1 kb in length), are heterogeneous in sequence, and encode gRNAs. The gRNAs are essential for the RNA editing process whereby the maxic...

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A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Ogbadoyi

Robinson

Gull

2003

MBoC

234

279

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“…In these organisms, the mitochondrial genome is highly unusual, comprising a mass of catenated DNA called the kinetoplast. Each cell has one kinetoplast such that replication and segregation of this organelle must be carefully controlled during the cell cycle (Klingbeil et al, 2001). The kinetoplast is composed of ϳ50 maxicircles (of ϳ23 kilobases [kb]) and several thousand 1-kb minicircles.…”

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confidence: 99%

Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

Timms

Deursen

Hendriks

et al. 2002

MBoC

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Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.

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“…[1][2][3]. The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each.…”

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Mitochondrial DNA ligase in Crithidia fasciculata

Sinha

Hines

Downey

2004

Proc. Natl. Acad. Sci. U.S.A.

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Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase ␤, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the ␤ polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).T he mitochondrial DNA of trypanosomatid protozoa (kinetoplast DNA, or kDNA) consists of two forms of topologically interlocked circular DNAs, minicircles, and maxicircles (for recent reviews of kDNA structure and replication, see refs. 1-3). The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each. The maxicircles encode genes for mitochondrial proteins and ribosomal RNAs. Minicircles encode small guide RNAs that serve as templates for RNA editing of maxicircle transcripts (4). In vivo, the kDNA exists as a condensed disk structure 1 m in diameter and Ϸ0.4 m thick. Each minicircle in the kDNA disk is linked to three other minicircles on average and is relaxed, unlike most other circular DNAs in nature, which are negatively supercoiled (5). Electron microscopic observation of sections through the kDNA disk shows fibers aligned parallel to the axis of the disk. This observation together with the thickness of the disk corresponding to approximately one half of the minicircle circumference (6) suggests that individual minicircles may have a topology like that of a rubber band stretched taut from opposite sides of the circle (7). Maxicircles appear to be present throughout the kDNA and exist as a catenated network within the overall network (8). The kDNA isolated from nonreplicating C. fasciculata is a planar elliptical network of interlocked circles with dimensions of Ϸ10 by 15 m.The kDNA disk is always located at the base of the flagellum, with the flagellum perpendicular to the face of the disk. Recent studies (9) have shown that in trypanosomes the kDNA disk is connected physically to the flagellar basal bodies and is segregated by them at cell division. The attachment was found to involve three components that penetrate the inner and outer mitochondrial membranes to link the kDNA physically ...

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Unlocking the Secrets of Trypanosome Kinetoplast DNA Network Replication

Cited by 49 publications

References 45 publications

A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

A High-OrderTrans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes

Mitochondrial Development during Life Cycle Differentiation of African Trypanosomes: Evidence for a Kinetoplast-dependent Differentiation Control Point

Mitochondrial DNA ligase in Crithidia fasciculata

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