Mitochondrial DNA ligase in Crithidia fasciculata

Abstract

Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure. A type II DNA topoisomerase, a DNA polymerase ␤, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles. We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk. DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA. The ligase interacts physically with the ␤ polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles. In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase. The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C. fasciculata nuclear DNA ligase (LIG I).T he mitochondrial DNA of trypanosomatid protozoa (kinetoplast DNA, or kDNA) consists of two forms of topologically interlocked circular DNAs, minicircles, and maxicircles (for recent reviews of kDNA structure and replication, see refs. 1-3). The kDNA of the trypanosomatid Crithidia fasciculata contains 5,000 minicircles of 2.5 kb each and 20-30 maxicircles of 37 kb each. The maxicircles encode genes for mitochondrial proteins and ribosomal RNAs. Minicircles encode small guide RNAs that serve as templates for RNA editing of maxicircle transcripts (4). In vivo, the kDNA exists as a condensed disk structure 1 m in diameter and Ϸ0.4 m thick. Each minicircle in the kDNA disk is linked to three other minicircles on average and is relaxed, unlike most other circular DNAs in nature, which are negatively supercoiled (5). Electron microscopic observation of sections through the kDNA disk shows fibers aligned parallel to the axis of the disk. This observation together with the thickness of the disk corresponding to approximately one half of the minicircle circumference (6) suggests that individual minicircles may have a topology like that of a rubber band stretched taut from opposite sides of the circle (7). Maxicircles appear to be present throughout the kDNA and exist as a catenated network within the overall network (8). The kDNA isolated from nonreplicating C. fasciculata is a planar elliptical network of interlocked circles with dimensions of Ϸ10 by 15 m.The kDNA disk is always located at the base of the flagellum, with the flagellum perpendicular to the face of the disk. Recent studies (9) have shown that in trypanosomes the kDNA disk is connected physically to the flagellar basal bodies and is segregated by them at cell division. The attachment was found to involve three components that penetrate the inner and outer mitochondrial membranes to link the kDNA physically ...

Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

“…This observation indicates that the MRB1 complex may have a distinct role in gRNA synthesis, since mtRNAP was not demonstrated to be associated with this complex in any of the published reports Hashimi et al 2008;Panigrahi et al 2008). Furthermore, the possibility that the mtRNAP transcribes both minicircle-encoded gRNAs and maxicircle-encoded mRNAs is surprising given the requirement of the multiple DNA ligases and DNA polymerases for kDNA maintenance and replication (Klingbeil et al 2002;Sinha et al 2004;Liu et al 2005).…”

Mitochondrial origin-binding protein UMSBP mediates DNA replication and segregation in trypanosomes

“…To study the status of gap-filling of minicircles in the giant kDNA networks, we have used the in situ incorporation of fluorescently labeled dUTP into kDNA networks by terminal deoxynucleotidyl transferase (TdT) (16). Under normal growth conditions, the final gap filling and sealing of replicated minicircles occurs after their reattachment to the kDNA network, possibly through the action of DNA polymerase ␤-PAK and ligase k␣ (17)(18)(19). Indeed, in uninduced cells, most of the kDNA networks were of an apparently normal size and were negative for the dUTP fluorescence signal, whereas larger kDNA networks display a fluorescence signal, indicating that they were replicating networks containing gapped minicircles (Fig.…”

A type II ribonuclease H from Leishmania mitochondria: An enzyme essential for the growth of the parasite

“…Thus, various enzymes involved in the kDNA replication are strategically placed around the kDNA disc. The DNA polymerase β [20], topoisomerase II [21], DNA ligase [23] and SSE1 [24] are localized in two antipodal sites, while the universal minicircle sequence binding protein [25] is found in the kinetoflagellar zone (KFZ). Primase was found to be on both the sides of the kDNA disc [26].…”