Closing the gaps in kinetoplast DNA network replication.

T rypanosomatids are protozoan parasites responsible for important tropical diseases. One example, Trypanosoma brucei, causes African sleeping sickness, and related parasites cause Chagas disease and leishmaniasis. Because they are among the earliest-branching eukaryotes, trypanosomatids have unusual biological properties. One of their most curious features is a unique mitochondrial DNA network known as kinetoplast DNA (kDNA) (1). The kDNA network is composed of several thousand minicircles that are interlocked like the links in medieval chain mail. Also intertwined in the network are a few dozen maxicircles. See Fig. 1 for an electron micrograph of a segment of an isolated kDNA network from Crithidia fasciculata, a trypanosomatid often studied because it is nonpathogenic and easy to cultivate. The function of kDNA maxicircles, like mitochondrial DNA in conventional eukaryotes, is to encode a few gene products such as rRNA and subunits of respiratory complexes. However, the mechanism of gene expression is highly unconventional in that maxicircle transcripts must be edited to form a functional mRNA. Editing is an amazing form of RNA processing in which uridine residues are inserted or deleted at precise internal sites within the maxicircle transcripts, generating ORFs. Minicircles encode guide RNAs that are templates for editing of maxicircle transcripts. See ref. 2 for a review of editing and ref. 3 for a discussion of the evolution of kDNA and the significance of the network structure. In this issue of PNAS, Sinha et al. (4) describe a novel C. fasciculata mitochondrial DNA ligase. This enzyme is distinct from the nuclear ligase I from the same organism (5) and from all other eukaryotic DNA ligases reported to date. Its small size (56 kDa) and sequence are reminiscent of virally encoded ligases. In yeast, a single gene encodes both a nuclear and a mitochondrial DNA ligase I by using an internal in-frame AUG to produce the nuclear protein (6). Vertebrates also produce a dual-targeted ligase (DNA ligase III) via a similar mechanism, and the mitochondrial version is present in two isoforms that differ at their C termini (reviewed in ref. 7). In contrast, the T. brucei genome encodes multiple ligases that appear to target specifically to the mitochondrion (4) (M. Lindsay and M.M.K., unpublished data) and which are likely involved in kDNA replication or repair. As discussed below, the novel intramitochondrial localization of the C. fasciculata ligase suggests a role in repairing gaps in newly replicated kDNA minicircles (e.g., joining Okazaki fragments) (4). Each trypanosomatid cell contains one kDNA network in the matrix of its single mitochondrion. The kDNA is