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.     

Kinetoplast DNA of Trypanosoma brucei: Physical map of the maxicircle

Trypanosoma brucei maxicircle DNA in kinetoplast DNA (kDNA) networks was characterized with restriction endonucleases. The data allow the construction of a circular map of a 22.2-kb molecule. Based on these and previous data each T. brucei kDNA network contains about 45 maxicircles which probably have the same sequence. The maxicircle of strain 164 used in this study was slightly larger and had three EcoRI sites compared to two found in other strains. Fragments generated by digestion with BamHI were largely singly cleaved maxicircles that had a density of 1.681 g/cm³ compared to 1.693 g/cm³ for the intact network. This suggests that maxicircles have a higher A + T content than minicircles. Minicircles in the kDNA network were also characterized with restriction endonucleases. Each enzyme cleaved a specific subset of minicircles from the network. However, no single restriction endonuclease or combination of up to three of these enzymes cleaved all molecules in the network. These results are consistent with earlier results of renaturation kinetic experiments and indicate that there are many different sequence classes of mini-circle DNA.     

Kinetoplast DNA of Bodo caudatus: a noncatenated structure.

The kinetoplast DNA (kDNA) of trypanosomes and other parasitic members of the order Kinetoplastida is organized as a complex network containing thousands of catenated circular DNA molecules. We found that the kDNA of a free-living kinetoplastida, Bodo caudatus, exists as a noncatenated structure. The kDNA of B. caudatus represents about 40% of the total cellular DNA, and the major components of this DNA are large circles of 10 and 12 kilobases (kb). Our results indicate that these circles are analogous to trypanosome kDNA minicircles despite their large size and noncatenated form. The kDNA of B. caudatus also contains a minor component of 19 kb which is transcribed. The 19-kb molecules are probably analogous to the maxicircles of trypanosomes. The properties of the B. caudatus kDNA suggest that the catenated network structure of trypanosome kDNA is not required for maxicircle segregation during kinetoplast division or for the expression of the maxicircle genome.     

Characterization of the molecular components in kinetoplast- mitochondrial DNA of Trypanosoma equiperdum. Comparative study of the dyskinetoplastic and wild strains

The structure of the kinetoplast DNA of Trypanosoma equiperdum has been studied and compared to the structure of the circular mitochondrial DNA extracted from a dyskinetoplastic strain of T. equiperdum. In T. equiperdum wild type, the kinetoplast DNA constitutes approximately 6% of the total cellular DNA and is composed of approximately 3,000 supercoiled minicircles of 6.4 x 10(5) daltons and approximately 50 circular supercoiled molecules of 15.4 x 10(6) daltons topologically interlocked; The buoyant density in CsCl of the minicircles is 1.691 g/cm 3. The large circles have a buoyant density of 1.684 g/cm 3, are homogeneous in size and are selectively cleaved by several restriction endonucleases which do not cleave the minicircles. The cleavage sites of six different restriction endonucleases have been mapped on the large circle. The minicircles are cleaved by two other restriction endonucleases, and their cleavage sites have been mapped. The mitochondrial DNA extracted from the dyskinetoplastic strain of T. equiperdum represents 7% of the total DNA of the cell and is composed of supercoiled circles, heterogeneous in size, and topologically associated in catenated oligomers. Its buoyant density in CsCl is 1.688 g/cm 3. These molecules are not cleaved by any of the eight restriction endonucleases tested. The reassociation kinetics of in vitro labeled kDNA minicircles and large circles has been studied. The results indicate that the minicircles as well as the large circles are homogeneous in sequence and that the circular DNA of the dyskinetoplastic strain has no sequence in common with the kDNA of the wild strain.     

Replication of kinetoplast DNA in isolated kinetoplasts from Crithidia fasciculata. Identification of minicircle DNA replication intermediates

The kinetoplast DNA (kDNA) of trypanosomes is comprised of thousands of DNA minicircles and 20-50 maxicircles catenated into a single network. We show that kinetoplasts isolated from the trypanosomatid species Crithidia fasciculata incorporate labeled nucleotides and support minicircle DNA replication in a manner which mimics two characteristics of minicircle replication in vivo: 1) the minicircles are replicated as free molecules and subsequently reattached to the kDNA network, and 2) a replication intermediate having a structure consistent with a highly gapped minicircle species is generated. In addition, a class of minicircle DNA replication intermediates is observed containing discontinuities at specific sites within each of the newly synthesized DNA strands. By using a strain of C. fasciculata possessing nearly homogenous minicircles, we were able to map the discontinuities to two small regions situated 180 degrees apart on the minicircle. Each region has two sites at which a discontinuity can occur, one on each strand and separated by approximately 100 base pairs. These sites may represent origins of minicircle DNA replication.     

Organized packaging of kinetoplast DNA networks

The kinetoplast DNA (kDNA) of Trypanosoma equiperdum is organized as a complex structure of catenated circular DNA molecules. The major component of the kDNA network is the one kilobase minicircle that is present at about 10,000 copies per network. We have developed two assays to examine the structure of kDNA networks compacted in vitro with spermidine. Our results suggest that minicircles are arranged into a regular structure with an exposed domain which is DNAase I- and restriction-sensitive and a protected domain which is resistant to restriction endonucleases and DNAase I. This regularly packaged structure is dependent upon spermidine compaction and the circularity of the kDNA, but does not require supercoiled minicircles or catenated networks.     

Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes

The unique mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles called kinetoplast DNA (kDNA). The network is essential for survival, and requires an elaborate topoisomerase‐mediated release and reattachment mechanism for minicircle theta structure replication. At least seven DNA polymerases (pols) are involved in kDNA transactions, including three essential proteins related to bacterial DNA pol I (POLIB, POLIC and POLID). How Trypanosoma brucei utilizes multiple DNA pols to complete the topologically complex task of kDNA replication is unknown. To fill this gap in knowledge we investigated the cellular role of POLIB using RNA interference (RNAi). POLIB silencing resulted in growth inhibition and progressive loss of kDNA networks. Additionally, unreplicated covalently closed precursors become the most abundant minicircle replication intermediate as minicircle copy number declines. Leading and lagging strand minicircle progeny similarly declined during POLIB silencing, indicating POLIB had no apparent strand preference. Interestingly, POLIB RNAi led to the accumulation of a novel population of free minicircles that is composed mainly of covalently closed minicircle dimers. Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery.     

Selection for arsenite resistance causes reversible changes in minicircle composition and kinetoplast organization in Leishmania mexicana.

Certain minor minicircle sequence classes in the kinetoplast DNA (kDNA) networks of arsenite- or tunicamycin-resistant Leishmania mexicana amazonensis variants whose nuclear DNA is amplified appear to be preferentially selected to replicate (S. T. Lee, C. Tarn, and K. P. Chang, Mol. Biochem. Parasitol. 58:187-204, 1993). These sequences replace the predominant wild-type minicircle sequences to become dominant species in the kDNA network. The switch from wild-type-specific to variant-specific minicircles takes place rapidly within the same network, the period of minicircle dominance changes being defined as the transition period. To investigate the structural organization of the kDNA networks during this transition period, we analyzed kDNA from whole arsenite-resistant Leishmania parasites by dot hybridization with sequence-specific DNA probes and by electron-microscopic examination of isolated kDNA networks in vitro. Both analyses concluded that during the switch of dominance the predominant wild-type minicircle class was rapidly lost and that selective replication of variant-specific minicircles subsequently filled the network step by step. There was a time during the transition when few wild-type- or variant-specific minicircles were present, leaving the network almost empty and exposing a species of thick, long, fibrous DNA which seemed to form a skeleton for the network. Both minicircles and maxicircles were found to attach to these long DNA fibrils. The nature of the long DNA fibrils is not clear, but they may be important in providing a framework for the network structure and a support for the replication of minicircles and maxicircles.     

The killing of African trypanosomes by ethidium bromide

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.     

Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol gamma) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.     

Pankinetoplast DNA structure in a primitive bodonid flagellate, Cryptobia helicis.

The mitochondrial DNA (mtDNA) of a primitive kinetoplastid flagellate Cryptobia helicis is composed of 4.2 kb minicircles and 43 kb maxicircles. 85% and 6% of the minicircles are in the form of supercoiled (SC) and relaxed (OC) monomers, respectively. The remaining minicircles (9%) constitute catenated oligomers composed of both the SC and OC molecules. Minicircles contain bent helix and sequences homologous to the minicircle conserved sequence blocks. Maxicircles encode typical mitochondrial genes and are not catenated. The mtDNA, which we describe with the term 'pankinetoplast DNA', is spread throughout the mitochondrial lumen, where it is associated with multiple electron-lucent loci. There are approximately 8400 minicircles per pankinetoplast-mitochondrion, with the pan-kDNA representing approximately 36% of the total cellular DNA. Based on the similarity of the C.helicis minicircles to plasmids, we present a theory on the formation of the kDNA network.     

TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments

Trypanosoma brucei''s mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei''s six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5′ to 3′ DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments.     

Mitochondrial DNA ligases of Trypanosoma brucei

The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k alpha and LIG k beta) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k alpha and LIG k beta are mitochondrial DNA ligases. Epitope-tagged LIG k alpha localizes throughout the kDNA, whereas LIG k beta shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k alpha mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.     

The kinetoplast DNA of the Australian trypanosome,Trypanosoma copemani,shares features with Trypanosoma cruzi and Trypanosoma lewisi

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10?bp sequence) and CSB-2 (8?bp sequence) present lower interspecies homology, while CSB-3 (12?bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257?bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.     

Kinetoplast DNA in Trypanosoma equinum1

Identification and characterization of kDNA is described in the naturally occurring totally dyskinetoplastic species Trypanosoma equinum. Fluorescence microscopy of live cells, using the highly sensitive and specific probe DAPI (4,6,-diamidino-2-phenyl-indole), showed the presence of a diversity of extranuclear fluorescent bodies scattered along the length of the organism. Transmission electron microscopic studies revealed a close similarity between the distribution of these DAPI-fluorescing particles and of dense aggregates of nonfibrillar material resembling the kDNA of dyskinetoplastic strains of other species. Variable sized remnants of kDNA, occurring singly or in clusters, were found scattered throughout the mitochondrion. Analytical cesium chloride ultracentrifugation of total cellular DNA extracts showed a kDNA banding profile at a buoyant density equal to 1.691 gm/cm³, representing approximately 11% of the total cellular DNA content. Molecular spreads of isolated kDNA revealed a population of open circular molecules ranging in contour length from 0.11–9.69 μm.     

The rotational dynamics of kinetoplast DNA replication

Kinetoplast DNA (kDNA), from trypanosomatid mitochondria, is a network containing several thousand catenated minicircles that is condensed into a disk-shaped structure in vivo. kDNA synthesis involves release of individual minicircles from the network, replication of the free minicircles and reattachment of progeny at two sites on the network periphery approximately 180 degrees apart. In Crithidia fasciculata, rotation of the kDNA disk relative to the antipodal attachment sites results in distribution of progeny minicircles in a ring around the network periphery. In contrast, Trypanosoma brucei progeny minicircles accumulate on opposite ends of the kDNA disk, a pattern that did not suggest kinetoplast motion. Thus, there seemed to be two distinct replication mechanisms. Based on fluorescence microscopy of the kDNA network undergoing replication, we now report that the T. brucei kinetoplast does move relative to the antipodal sites. Whereas the C. fasciculata kinetoplast rotates, that from T. brucei oscillates. Kinetoplast motion of either type must facilitate orderly replication of this incredibly complex structure.