The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata

Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.     

Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids.

Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms. The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells. Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles. The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence. As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S. cerevisiae.     

A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization

Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.     

Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.     

Visualization of the bent helix in kinetoplast DNA by electron microscopy

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.     

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.     

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.     

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Cationic metals promote sequence-directed DNA bending

A DNA segment of approximately 200 base pairs (bp) from Crithidia fasciculata kinetoplast minicircles was previously shown by electron microscopy (EM) to bend into a small circle due to its unique nucleotide sequence containing repeated blocks of 4-6 A's. When this segment was flanked by 207 bp of plasmid DNA on one side and 460 bp on the other, the resulting 890-bp DNA was found to appear either relatively straight or extremely bent as visualized by EM. The bend was located one-third the distance from one end. The fraction of molecules with the most extreme bend increased from approximately 2% to 50-60% following incubation of the DNA with increasing concentrations of Zn2+, Co2+, Ba2+, and Mn2+. These observations suggest that sequence-directed bending in DNA is an inducible and not a static phenomenon. Possible roles of transitions between the bent and straight conformations in the control of gene expression are discussed.     

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.     

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.     

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.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.     

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.     

Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I.

DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.     

Kinetoplast DNA minicircles of trypanosomatids encode for a protein product

The major constituent of the trypanosomal kinetoplast DNA network are several thousand duplex DNA minicircles whose biological function is still unknown. The coding capacity and expression of these DNA minicircles, was studied in the trypanosomatid Crithidia fasciculata. Kinetoplast DNA minicircle fragments inserted into bacterial plasmid vectors were expressed in the bacterial cell. Sera elicited in rabbits, by immunization with the translational products of kinetoplast DNA minicircles in E. coli, reacted specifically with Crithidia fasciculata cellular antigens. It is inferred that kinetoplast DNA minicircles contain long open reading frames of nucleotides which are expressed in the trypanosomatid cell.     

Ribonucleotides associated with a gap in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum

In Trypanosoma equiperdum, some newly replicated kinetoplast DNA minicircles contain a single gap at a unique location in their newly synthesized strand (Ntambi, J. M., and Englund, P. T. (1985) J. Biol. Chem. 260, 5574-5579). We now report that ribonucleotides are associated with this gap, with one or two covalently attached to the 5' terminus of the newly synthesized strand. There appear to be two possible RNA/DNA junctions at adjacent positions in the sequence. The ribonucleotides may be remnants of a replication primer, and their presence strongly implies that the gap is at the site of a replication origin.     

Universal minicircle sequence-binding protein, a sequence-specific DNA-binding protein that recognizes the two replication origins of the kinetoplast DNA minicircle

Replication of the kinetoplast DNA minicircle lagging (heavy (H))-strand initiates at, or near, a unique hexameric sequence (5'-ACGCCC-3') that is conserved in the minicircles of trypanosomatid species. A protein from the trypanosomatid Crithidia fasciculata binds specifically a 14-mer sequence, consisting of the complementary strand hexamer and eight flanking nucleotides at the H-strand replication origin. This protein was identified as the previously described universal minicircle sequence (UMS)-binding protein (UMSBP) (Tzfati, Y., Abeliovich, H., Avrahami, D., and Shlomai, J. (1995) J. Biol. Chem. 270, 21339-21345). This CCHC-type zinc finger protein binds the single-stranded form of both the 12-mer (UMS) and 14-mer sequences, at the replication origins of the minicircle L-strand and H-strand, respectively. The attribution of the two different DNA binding activities to the same protein relies on their co-purification from C. fasciculata cell extracts and on the high affinity of recombinant UMSBP to the two origin-associated sequences. Both the conserved H-strand hexamer and its flanking nucleotides at the replication origin are required for binding. Neither the hexameric sequence per se nor this sequence flanked by different sequences could support the generation of specific nucleoprotein complexes. Stoichiometry analysis indicates that each UMSBP molecule binds either of the two origin-associated sequences in the nucleoprotein complex but not both simultaneously.     

Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation.

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.     

kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.