Drug-promoted cleavage of kinetoplast DNA minicircles. Evidence for type II topoisomerase activity in trypanosome mitochondria.

Minicircle DNA, the major component of the mitochondrial DNA of trypanosomes (kinetoplast DNA), is linearized when living Trypanosoma equiperdum cells are treated with inhibitors of mammalian type II topoisomerases and then lysed with sodium dodecyl sulfate. A variety of intercalating and nonintercalating compounds (the epipodophyllotoxins, 4'-(9-acridinylamino)-methanesulfon-m-anisidine, 2-methyl-9-hydroxyellipticine, and acriflavine) are active, but novobiocin and specific gyrase inhibitors (the quinolones) are not. The linearized minicircles are in a DNA-protein complex, as their electrophoretic mobility is increased by Proteinase K treatment. They are digested by exonuclease III but not by lambda exonuclease, indicating that the protein must be linked to both 5' ends. Drug-induced cleavage sites vary with different compounds and are found throughout the minicircle sequence. These results indicate that trypanosome mitochondria contain a type II topoisomerase with some properties similar to those of type II topoisomerases in the nucleus of higher eukaryotes. A maximum of 12% of all minicircles is cleaved in the presence of VP16-213, indicating there are at least 600 molecules of mitochondrial type II topoisomerase/cell or about one enzyme/8 kilobases of minicircle DNA.

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.

Intermediates in the replication of kinetoplast DNA minicircles.

Kinetoplast DNA of Crithidia fasciculata and other trypanosomatids is in the form of a network of thousands of minicircles and a few dozen maxicircles. Minicircles replicate as free molecules after release from the network, and their progeny subsequently reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). The minicircles just released from the network are covalently closed and apparently completely relaxed. After Cairns-type (theta) replication, the two minicircle progeny have different structures. One has a nascent H (heavy) strand which initially is in the form of 20-110 nucleotide fragments that are separated by gaps (Kitchin, P. A., Klein, V. A., Fein, B. I., and Englund, P. T. (1984) J. Biol. Chem. 259, 15532-15539). The other initially has a full-size (2.5 kilobase) nascent L (light) strand. During the time between formation of these progeny molecules and network reattachment, the nascent L strand is nicked (or gapped) and nascent H strand is partially repaired. Therefore, both progeny, at the time of reattachment, have several nicks (or gaps) in their nascent strand. Minicircle progeny with a nascent L strand reattach to the network quickly, whereas those with a nascent H strand reattach more slowly. Once reattached to the network, the nicks or gaps in the minicircles are repaired until finally covalent closure occurs.

Gapped Minicircles. A novel replication intermediate of kinetoplast DNA.

Kinetoplast DNA, the mitochondrial DNA in trypanosomatids, is a network of thousands of interlocked circles. Most of these circles are minicircles and a few are maxicircles. Minicircles replicate, after decatenation from the network, by a Cairns-type mechanism. The minicircle progeny then reattach to the network (Englund, P. T. (1979) J. Biol. Chem. 254, 4895-4900). We have now discovered a novel intermediate in Crithidia fasciculata minicircle replication. It is a highly gapped 2.5-kilobase free minicircle with nascent fragments of only 20 to 110 nucleotides. These fragments are nonligasable, and some remain nonligasable even after gap filling with DNA polymerase. Solution hybridization studies show that the nascent fragments are predominantly, if not exclusively, heavy strand.

Presence of a bent helix in fragments of kinetoplast DNA minicircles from several trypanosomatid species.

Some restriction fragments of kinetoplast minicircles from several trypanosomatid species (Leishmania tarentolae, Trypanosoma brucei, T. equiperdum, Herpetomonas muscarum, Crithidia fasciculata, but not T. cruzi) migrate anomalously on polyacrylamide gels. This behavior is probably due to a natural curvature of the helix. Bent helices appear to be a common property of kinetoplast minicircles, and may be important for minicircle function. In the case of T. equiperdum, we present evidence that each minicircle has a single bent region which resides in or near the 'conserved sequence.'

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.