No abstract

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His 6 -tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.The mitochondrial DNA of kinetoplastid protozoa consists of about 5,000 minicircles and 20 to 30 maxicircles. These circular DNAs are held together by catenation into a single two-dimensional sheet of DNA referred to as a kinetoplast DNA (kDNA) network. The minicircles exist within the network as relaxed covalently closed circles, with each minicircle linked on average to three other minicircles (3). Each cell has only one such network, which in purified form or in cell lysates has a diameter similar in size (8 to 10 m) to that of the whole cell. In vivo, the kDNA network exists as a highly condensed disc about 1 m in diameter and approximately 0.4 m thick (25; also see below). The kDNA disc is physically associated with the basal body of the cell and is oriented with the axis of the disc parallel to that of the flagellum. Electron micrographs of sections through the kDNA disc also show DNA fibers oriented parallel to the axis of the disc. For recent reviews of the structure and replication of kinetoplast DNA, see references 7 and 24.We have developed methods recently for identifying and characterizing proteins that may play a role in organizing and condensing the kDNA network into the compact disc structure observed in vivo (35). Proteins bound to kDNA are covalently...

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␣ and LIG k␤) 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␣ and LIG k␤ are mitochondrial DNA ligases. Epitope-tagged LIG k␣ localizes throughout the kDNA, whereas LIG k␤ 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␣ 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.

Kinetoplast DNA minicircles from various species of trypanosomes are heterogeneous in nucleotide sequence to various degrees but in all instances contain a conserved sequence region of 100 to 200 base pairs present in one, two, or four copies per minicircle. Comparison of the conserved sequence regions of minicircies from eight species of trypanosomes revealed a common sequence motif consisting of three conserved sequence blocks (CSBs) present in the same order and with similar spacing in all species. In addition to the invariant 12-base-pair universal minicircle sequence (CSB-3), a 10-base-pair sequence (CSB-1) and an 8-base-pair sequence (CSB-2) are highly conserved in all minicircies. The overlap of CSB-1 and CSB-3 with previously identified 5' termini of newly synthesized minicircle H and L strands, respectively, and the presence of this conserved sequence motif in minicircles from diverse species suggest that these CSBs may determine a common mechanism of mjnicircle replication.

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Gene expression in trypanosomatids appears to be regulated largely at the posttranscriptional level and involves maturation of mRNA precursors by trans splicing of a 39-nucleotide miniexon sequence to the 5' end of the mRNA and cleavage and polyadenylation at the 3' end of the mRNA. To initiate the identification of sequences involved in the periodic expression of DNA replication genes in trypanosomatids, we have mapped splice acceptor sites in the 5' flanking region of the TOP2 gene, which encodes the kinetoplast DNA topoisomerase, and have carried out deletion analysis of this region on a plasmid-encoded TOP2 gene. Block deletions within the 5' untranslated region (UTR) identified two regions (-608 to -388 and -387 to -186) responsible for periodic accumulation of the mRNA. Deletion of one or the other of these sequences had no effect on periodic expression of the mRNA, while deletion of both regions resulted in constitutive expression of the mRNA throughout the cell cycle. Subcloning of these sequences into the 5' UTR of a construct lacking both regions of the TOP2 5' UTR has shown that an octamer consensus sequence present in the 5' UTR of the TOP2, RPA1, and DHFR-TS mRNAs is required for normal cycling of the TOP2 mRNA. Mutation of the consensus octamer sequence in the TOP2 5' UTR in a plasmid construct containing only a single consensus octamer and that shows normal cycling of the plasmid-encoded TOP2 mRNA resulted in substantial reduction of the cycling of the mRNA level. These results imply a negative regulation of TOP2 mRNA during the cell cycle by a mechanism involving redundant elements containing one or more copies of a conserved octamer sequence within the 5' UTR of TOP2 mRNA.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the f lagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5 exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase ␤ at antipodal sites f lanking the kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell The trypanosomatid Crithidia fasciculata has an unusual mitochondrial DNA structure called the kinetoplast DNA, which is composed of 5,000 minicircles of 2.5 kilobases (kb) and 25 maxicircles of 37 kb catenated into a single network. The maxicircles encode conventional mitochondrial proteins and ribosomal RNAs. Unlike most mitochondrial DNA, kinetoplast DNA replicates only once in the cell cycle in approximate synchrony with nuclear DNA replication (1, 2). Minicircles replicate free of the network as intermediates (3). Lightstrand synthesis is RNA primed at each of the two universal minicircle sequences and occurs continuously (4-6). Heavystrand synthesis occurs discontinuously via Okazaki-like fragments that are also likely to be RNA primed (5, 7). Nicks and gaps remaining in newly replicated minicircles are partially repaired before reattachment to the network but are not completely repaired until just before network division into two daughter networks. (4,5,8,9).Several enzymes involved in kinetoplast DNA replication have been purified and localized within the kinetoplast. A primase has been localized to a region adjacent to the two faces of the kinetoplast disk, suggesting that RNA priming and the early stages of minicircle replication may occur in this region (10). Additionally, a topoisomerase II and a DNA polymerase ␤ have been localized to two antipodal sites flanking the kinetoplast disc where the minicircles are reattached to the network (11-14). Minicircle replication intermediates have also been detected at these sites (11). The low processivity of polymerase ␤ suggests that it may be involved in the repair of gaps remaining in newly replicated minicircles. The topoisomerase II is likely to mediate minicircle reattachment after partial gap repair.To further define the components of the replication machinery and their localization relative to the kinetoplast disc, we have sought to identify and purify additional kinetoplast replication proteins. The purification and characterization of a structure-specific endonuclease (SSE1) enriched in the kinetoplast fraction of C. fasciculata ...

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is a highly condensed disc-shaped network of catenated DNA cirdes consisting of maxicircdes, the equivalent of conventional mitochondrial DNA, and several thousand smaller circular DNAs termed minicircies. Upon cell lysis, kDNA expands, giving rise to a two-dimensional network of catenated circles with an overall diameter dose to that of the whole cell. To identify proteins associated with the condensed form of kDNA in the cell, proteins were reversibly crosslinked to kDNA in whole cells of Crithidiafasciculata by formaldehyde treatment. Crosslinked networks were purified and found to retain a condensed structure which becomes fufly expanded upon proteinase K treatment or reversal of the crosslinks by heating at 65C. Five low molecular weight proteins released from the kDNA by heat treatment were purified by polyacrylamide gel electrophoresis and their amino-terminal sequences were determined. PCR amplification and sequence analysis of cDNA sequences between these amino-terminal sequences and the miniexon (spliced leader) sequence present at the 5' end of all C.fascicula mRNAs predicts the presence of 9-amino acid presequences with features characteristic of mitochondrial presequences on three ofthe proteins. Two ofthese proteins are lysine-rich basic proteins. These rmdings suggest that basic proteins may play a role in the condensation of kDNA in the kinetoplast and that these proteins are imported into the kinetoplast by a mechanism involving a cleavable presequence.