Repeated sequences with unusual properties from the divergent region of Crithidia oncopelti maxicircle kinetoplast DNA

Summary The occurrence of bacterial promoter-like sequences was shown in the divergent region ofCrithidia oncopelti maxicircular kinetoplast DNA. A promoter-cloning vector based on the neomycin phosphotransferase II (NPT II) gene was used to localize promoters in three homologous blocks of repeated sequences. The elements found displayed greater promoter strength inEscherichia coli than the normal promoter of the NPT II gene. Sequences of three promoter-containing inserts from different blocks were determined and typical prokaryotic promoter sequences were localized.     

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.     

Isolation and characterization of kinetoplast DNA from Leishmania tarentolae

Kinetoplast DNA (? = 1.703 g/ml.) was isolated by preparative cesium chloride ultracentrifugation in a fixed-angle rotor from total cell DNA of Leishmania tarentolae and examined in terms of sedimentation properties, melting characteristics, and appearance in the electron microscope. It consisted of several molecular types, either free or bound together in associations of variable size: minicircles (molecular weight = 0.56 ± 0.03 × 10⁶), catenated minicircles, “figure 8” molecules, and long molecules. The associations seem to be held together by the long molecules threading through the smaller circles and catenanes. The large associations could be broken down by sonication, DNase II-treatment, or shear forces. Minicircles, catenated dimers, trimers, and small linear fragments were separated on preparative sucrose gradients of sonicated DNA, and S_20,w values were assigned to each molecular type by band sedimentation in the analytical ultracentrifuge.     

The unusual conformation adopted by the adenine tracts in kinetoplast DNA

To determine the structural features responsible for the curvature of kinetoplast DNA, we studied 13 adenine tracts in Crithidia fasciculata kinetoplast DNA. The structures of the A tracts were analyzed by cutting the DNA with hydroxyl radical. Reactivity of hydroxyl radical toward the DNA backbone progressively decreased in the 5'----3' direction of each A tract. The cutting pattern of the T-rich strand was offset by 1 or 2 bp from the pattern on the A-rich strand. An A tract in a restriction fragment from plasmid pBR322 had the same cutting pattern as the kinetoplast A tracts. We interpret these experiments to show that in A tracts the width of the minor groove decreases smoothly from the 5'----3' end of the A tract.     

Physical properties of a genomic condensed chromatin fragment

We have studied the physical properties of a segment of condensed chromatin that lies upstream of the chicken beta-globin locus. This segment can be excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus as an essentially homogeneous fragment about 15.5 kbp long. Because of this homogeneity we could measure its sedimentation coefficient quite accurately by a combination of sucrose gradient and analytical ultracentrifugation. By measuring additionally the buoyant density of the cross-linked particle in CsCl we could deduce the total mass of the particle, hence its frictional coefficient, f, directly related to its shape. The measured value of f is consistent with a rod-like particle of the approximate length and diameter proposed earlier for the 30 nm chromatin fiber. The method is generally applicable to homogeneous particles of unique sequence at genomic abundance.     

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.     

Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei

We have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasis Research Organization [EATRO] 427). Endonuclease PstI and S1 nuclease cut the maxi-circle at a single site, allowing its isolation in a linear form with a mol wt of 12.2 x 10(6), determined by electron microscopy. The other enzymes give multiple maxi-circle fragments, whose added mol wt is 12-13 x 10(6), determined by gel electrophoresis. The maxi-circle in another T. brucei isolate (EATRO 1125) yields similar fragments but appears to contain a deletion of about 0.7 x 10(6) daltons. Electron microscopy of kDNA shows the presence of DNA considerably longer than the mini-circle contour length (0.3 micron) either in the network or as loops extending from the edge. This long DNA never exceeds the maxi-circle length (6.3 microns) and is completely removed by digestion with endonuclease PstI. 5-10% of the networks are doublets with up to 40 loops of DNA clustered between the two halves of the mini-circle network and probably represent a division stage of the kDNA. Digestion with PstI selectively removes these loops without markedly altering the mini-circle network. We conclude that the long DNA in both single and double networks represents maxi-circles and that long tandemly repeated oligomers of mini-circles are (virtually) absent. kDNA from Trypanosoma equiperdum, a trypanosome species incapable of synthesizing a fully functional mitochondrion, contains single and double networks of dimensions similar to those from T. brucei but without any DNA longer than mini-circle contour length. We conclude that the maxi-circle of trypanosomes is the genetic equivalent of the mitochondrial DNA (mtDNA) of other organisms.     

The assembly of kinetoplast DNA

The unusual structure of the kinetoplast DNA (kDNA) of trypanosomatids requires unique replication mechanisms. Deciphering the mechanisms that regulate the network assembly has been a challenge for many years. A better understanding of these processes was facilitated by recent studies on the fine structure of resting and replicating kDNA networks. In this review, Joseph Shlomai discusses our current view of the structural and mechanistic aspects of the assembly of kinetoplast DNA.     

Replication of kinetoplast DNA maxicircles

The kinetoplast DNA of Crithidia fasciculata is a massive network composed of thousands of topologically interlocked circles. Most of these circles are minicircles (2.5 kb), and about 50 are maxicircles (37 kb). Previous studies showed that minicircles replicate, after release from the network, via Cairns (theta) intermediates. Here we show that maxicircles replicate, while attached to the network, by an entirely different mechanism involving rolling circle intermediates. After the network-bound maxicircle has finished replication, the branch of the rolling circle is apparently cleaved off to form a linear free maxicircle. A restriction map of the linearized free maxicircles shows that these molecules have unique termini, one of which presumably corresponds to the replication origin.     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

The kinetoplast DNA of Trypanosoma equiperdum

We have analyzed the kinetoplast DNA for Trypanosoma equiperdum (American Type Culture Collection 30019) and two dyskinetoplastic strains derived from it. The DNA networks from the kinetoplastic strain are made up of catenated mini-circles and maxi-circles, like the networks from the closely-related Trypanosoma brucei. The mini-circles of T. equiperdum lack the pronounced sequence heterogeneity of T. brucei mini-circles, as shown by the fragment distribution of restriction digests and by the predominance of well-matched duplexes in electron micrographs of renatured DNA. The electrophoretic analysis of kinetoplast DNA digested with various restriction endonucleases shows the maxi-circle of T. equiperdum to consist of circular DNA molecules of 8.4 x 10(6) daltons, without size or sequence heterogeneity or repetitious segments. A comparison of the sequence by restriction endonuclease fragmentation and hybridization shows extensive sequence homology. The size difference between both maxi-circles is due to the deletion of one continuous segment of 5.10(6) daltons. In the two dyskinetoplastic strains, we cannot detect DNA sequences that hybridize with kinetoplast DNA from T. brucei or from the kinetoplastic strain of T. equiperdum. In one of these strains, a 'low-density' DNA fraction contained a simple sequence DNA, cleaved by restriction endonuclease HindIII into fragments of 180 base-pairs and multimers of this. The relation of this DNA to kinetoplast DNA, if any, is unknown.     

Reversible decatenation of kinetoplast DNA by a DNA topoisomerase from trypanosomatids.

DNA topoisomerase activity detected in cell extracts of the trypanosomatid Crithidia fasciculata interlocks kinetoplast DNA duplex minicircles into huge catenane forms resembling the natural kinetoplast DNA networks found in trypanosomes. Catenation of duplex DNA circles is reversible and equilibrium is affected by ionic strength, and by spermidine. The reaction requires magnesium, is ATP dependent and is inhibited by high concentrations of novobiocin. Extensive homology between duplex DNA rings was not required for catenane formation since DNA circles with unrelated sequences could be interlocked into mixed network forms. Covalently sealed catenaned DNA circles are specifically used as substrates for decatenation. No such preference for covalently sealed duplex DNA rings was observed for catenate formation. Its catalytic properties and DNA substrate preference, suggest a potential role for this eukaryotic topoisomerase activity in the replication of kinetoplast DNA.     

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.