Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.     

Isolation and Characterization of Kinetoplast DNA Networks and Minicircles from Crithidia fasciculata*

SYNOPSIS. Covalently closed kinetoplast DNA networks have been isolated from stationary phase Crithidia fasciculata cells by a technic involving selective pelleting of the networks at a low centrifugal field. Approximately 62% of the kinetoplast DNA of the cell was recovered free of nuclear DNA by simple differential centrifugation. Purified kinetoplast DNA networks were visualized both in the electron microscope and in the light microscope. Closed networks sedimented as a homogeneous band both in neutral and alkaline sucrose, with an s_20w in neutral sucrose of approximately 5 × 10³. Closed monomeric minicircles were isolated from purified networks by mild sonication and band sedimentation in alkaline sucrose. Several physical properties of closed monomeric minicircles were measured. These included molecular weight, buoyant density in CsCl, superhelix density and sedimentation coefficient.     

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.     

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.     

Characteristics of cytidine aminohydrolase activity in Trypanosoma cruzi and Crithidia fasciculata

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Intramitochondrial location and dynamics of Crithidia fasciculata kinetoplast minicircle replication intermediates

Kinetoplast DNA, the mitochondrial DNA of Crithidia fasciculata, is organized into a network containing 5,000 topologically interlocked minicircles. This network, situated within the mitochondrial matrix, is condensed into a disk-shaped structure located near the basal body of the flagellum. Fluorescence in situ hybridization revealed that before their replication, minicircles are released vectorially from the network face nearest the flagellum. Replication initiates in the zone between the flagellar face of the disk and the mitochondrial membrane (we term this region the kinetoflagellar zone [KFZ]). The replicating minicircles then move to two antipodal sites that flank the disk-shaped network. In later stages of replication, the number of free minicircles increases, accumulating transiently in the KFZ. The final replication events, including primer removal, repair of many of the gaps, and reattachment of the progeny minicircles to the network periphery, are thought to take place within the antipodal sites.     

Structural characterization of site-specific discontinuities associated with replication origins of minicircle DNA from Crithidia fasciculata

The kinetoplast DNA of trypanosomes is comprised of thousands of DNA minicircles and 20-50 maxicircles catenated into a single network. Replication intermediates of minicircle DNA from the trypanosomatid species Crithidia fasciculata contain site-specific discontinuities in both heavy (H) and light (L) strands. These discontinuities map 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. We have determined the position of these discontinuities on the minicircle DNA sequence and have characterized their structure. H-strand discontinuities occur within a 4-5-nucleotide sequence and consist of single nicks, only one of which appears to be a DNA-DNA junction. Characterization of the remaining H-strand nicks indicates a structure other than a typical DNA-DNA or DNA-RNA junction. Discontinuities on the L-strand can be either a nick or a short gap which overlaps a 12-nucleotide sequence universally conserved among minicircles from various trypanosome species. Up to 6 nucleotides are hydrolyzed from the 5' terminus facing the gap upon treatment with alkali, suggesting the presence of an RNA primer. Based on the structures of minicircle replication intermediates, we present a model for replication of minicircle DNA in which the site-specific discontinuities closely coincide with the origins of replication.     

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 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae

A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata. Chimeric plasmids containing this autonomously replicating sequence (ars) are maintained as extrachromosomal elements in S. cerevisiae. The nucleotide sequence of the maxicircle fragment, termed ars189, has been determined, and its position has been mapped in the maxicircle. The ars189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides. The ars189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C. fasciculata maxicircle genome. This sequence also shows extensive homology to a sequence within a 2.6-kb ars fragment of the Leishmania tarentolae maxicircle. In addition, ars189 contains two copies of a yeast consensus ars sequence (A/T)TTTATPuTTT(T/A).     

Characteristics of Cytidine Aminohydrolase Activity in Trypanosoma cruzi and Crithidia fasciculata1

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.     

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.     

The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast

The kinetoplast is a concatenated network of circular DNA molecules found in the mitochondrion of many trypanosomes. This mass of DNA is replicated in a discrete "S" phase in the cell cycle. We have tracked the incorporation of the thymidine analogue 5-bromodeoxyuridine into newly replicated DNA by immunofluorescence and novel immunogold labeling procedures. This has allowed the detection of particular sites of replicated DNA in the replicating and segregating kinetoplast. These studies provide a new method for observing kinetoplast DNA (kDNA) replication patterns at high resolution. The techniques reveal that initially the pattern of replicated DNA is antipodal and can be detected both on isolated complexes and in replicating kDNA in vivo. In Trypanosoma brucei the opposing edges of replicating kDNA never extend around the complete circumference of the network, as seen in other kinetoplastids. Furthermore, crescent-shaped labeling patterns are formed which give way to labeling of most of the replicating kDNA except the characteristic midzone. The configuration of these sites of replicated DNA molecules is different to previous studies on organisms such as Crithidia fasciculata, suggesting differences in the timing of replication of mini and maxicircles and/or organization of the replicative apparatus in the kinetoplast of the African trypanosome.     

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.     

A highly bent fragment of Crithidia fasciculata kinetoplast DNA

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.