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.     

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.     

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.     

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.     

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.     

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.     

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.     

The structure of replicating kinetoplast DNA networks

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.     

Nucleus-encoded histone H1-like proteins are associated with kinetoplast DNA in the trypanosomatid Crithidia fasciculata.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.     

Orientation of DNA Minicircles Balances Density and Topological Complexity in Kinetoplast DNA

Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network.     

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.     

Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID

Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.     

RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA

We studied the function of a Trypanosoma brucei topoisomerase II using RNA interference (RNAi). Expression of a topoisomerase II double-stranded RNA as a stem-loop caused specific degradation of mRNA followed by loss of protein. After 6 days of RNAi, the parasites' growth rate declined and the cells subsequently died. The most striking phenotype upon induction of RNAi was the loss of kinetoplast DNA (kDNA), the cell's catenated mitochondrial DNA network. The loss of kDNA was preceded by gradual shrinkage of the network and accumulation of gapped free minicircle replication intermediates. These facts, together with the localization of the enzyme in two antipodal sites flanking the kDNA, show that a function of this topoisomerase II is to attach free minicircles to the network periphery following their replication.     

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.     

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 minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.