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.