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.     

Overexpression of a cytochrome b5 reductase-like protein causes kinetoplast DNA loss in Trypanosoma brucei

The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.     

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.     

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.     

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.     

Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication

ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.     

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.     

A second mitochondrial DNA primase is essential for cell growth and kinetoplast minicircle DNA replication in Trypanosoma brucei

The mitochondrial DNA of trypanosomes contains two types of circular DNAs, minicircles and maxicircles. Both minicircles and maxicircles replicate from specific replication origins by unidirectional theta-type intermediates. Initiation of the minicircle leading strand and also that of at least the first Okazaki fragment involve RNA priming. The Trypanosoma brucei genome encodes two mitochondrial DNA primases, PRI1 and PRI2, related to the primases of eukaryotic nucleocytoplasmic large DNA viruses. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2ΔNT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2ΔNTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.     

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.     

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.     

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.     

Interactions of a replication initiator with histone H1-like proteins remodel the condensed mitochondrial genome

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, consists of several thousand topologically interlocked DNA circles. Mitochondrial histone H1-like proteins were implicated in the condensation of kDNA into a nucleoid structure in the mitochondrial matrix. However, the mechanism that remodels kDNA, promoting its accessibility to the replication machinery, has not yet been described. Analyses, using yeast two hybrid system, co-immunoprecipitation, and protein-protein cross-linking, revealed specific protein-protein interactions between the kDNA replication initiator protein universal minicircle sequence-binding protein (UMSBP) and two mitochondrial histone H1-like proteins. Fluorescence and electron microscopy, as well as biochemical analyses, demonstrated that these protein-protein interactions result in the decondensation of kDNA. UMSBP-mediated decondensation rendered the kDNA network accessible to topological decatenation by topoisomerase II, yielding free kDNA minicircle monomers. Hence, UMSBP has the potential capacity to function in vivo in the activation of the prereplication release of minicircles from the network, a key step in kDNA replication, which precedes and enables its replication initiation. These observations demonstrate the prereplication remodeling of a condensed mitochondrial DNA, which is mediated via specific interactions of histone-like proteins with a replication initiator, rather than through their posttranslational covalent modifications.     

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.     

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.     

TbPIF1, a Trypanosoma brucei Mitochondrial DNA Helicase, Is Essential for Kinetoplast Minicircle Replication

Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.