Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.     

Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.     

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.     

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.     

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.     

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.     

Effects of RNA interference of Trypanosoma brucei structure-specific endonuclease-I on kinetoplast DNA replication

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid protozoa, is a network containing several thousand topologically interlocked DNA minicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles, and reattachment of the progeny back onto the network. One enzyme involved in this process is structure-specific endonuclease-I. This enzyme, originally purified from Crithidia fasciculata, has been proposed to remove minicircle replication primers (Engel, M. L., and Ray, D. S. (1998) Nucleic Acids Res. 26, 4773-4778). We have studied the structure-specific endonuclease-I homolog from Trypanosoma brucei, showing it to be localized in the antipodal sites flanking the kinetoplast DNA disk, as previously shown in C. fasciculata. RNA interference of structure-specific endonuclease-I caused persistence of a single ribonucleotide at the 5' end of both the leading strand and at least the first Okazaki fragment in network minicircles, demonstrating that this enzyme in fact functions in primer removal. Probably because of the persistence of primers, RNA interference also impeded the reattachment of newly replicated free minicircles to the network and caused a delay in kinetoplast DNA segregation. These effects ultimately led to shrinkage and loss of the kinetoplast DNA network and cessation of growth of the cell.     

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.     

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.     

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.     

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.     

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.     

The replication of kinetoplast DNA networks in Crithidia fasciculata

Kinetoplast DNA from the mitochondria of Crithidia is in the form of a two-dimensional network of thousands of minicircles each containing about 2.5 kb, and a small number of maxicircles each containing about 40 kb. Fractionation of kinetoplast DNA by equilibrium centrifugation in a CsCl-propidium dilodide gradient resolves it into three types of networks. Form I networks band at high density and contain minicircles which are covalently closed; form II networks band at low density and contain minicircles which are nicked or gapped; and replicating networks band at intermediate density and contain some minicircles of each type. Form I networks contain about 5000 minicircles; form II networks contain about 11,000; and replicating networks contain an intermediate number. When cells are pulse-labeled with ³H-thymidine, radioactivity in mitochondrial DNA is preferentially incorporated into replicating networks, but after a chase it appears first in form II networks and finally in form I. Examination of replicating networks by electron microscopy in the presence of ethidium bromide reveals that minicircles in the central region of the network are twisted and therefore covalently closed, whereas those in the peripheral region are not twisted and therefore must be nicked or gapped. The pulse-label is incorporated into the nicked or gapped minicircles of the replicating networks. These results indicate that replication of form I networks begins in peripheral minicircles and that progeny minicircles remain nicked or gapped. As replication proceeds, the size of the network increases, and the peripheral zone of nicked or gapped minicircles enlarges. Finally, when all minicircles have replicated, the network, now form II, is double the size of form I and contains only nicked or gapped minicircles. The final step in replication presumably includes both the cleavage of the network into two form I species and the covalent closure of all the minicircles.     

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.     

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.     

Kinetoplast DNA minicircles: High-copy-number mitochondrial plasmids

The kinetoplast DNA of trypanosomes is a highly unusual network of catenated DNA circles of two kinds: maxicircles, the equivalent of conventional mitochondrial DNA, and minicircles, high-copy-number mitochondrial plasmids with no known function. Kinetoplast minicircles share many features with bacterial plasmids and represent a novel model system for the study of the mechanisms and regulation of DNA replication in eukaryotic organisms.