kDNA Replication Research Articles

RNAi libraries and kinetoplast DNA.

African trypanosomes have a remarkable mitochondrial DNA termed kDNA (kinetoplast DNA) that contains several thousands of topologically interlocked DNA rings. Because of its highly unusual structure, kDNA has a complex replication mechanism. Our approach to understanding this mechanism is to identify the proteins involved and to characterize their function. So far approx. 30 candidate proteins have been discovered and we predict that there are over 100. To identify genes for more kDNA replication proteins, we are using an RNA interference library, which is the first forward genetic approach used for these parasites.

RNAi libraries and kinetoplast DNA

African trypanosomes have a remarkable mitochondrial DNA termed kDNA (kinetoplast DNA) that contains several thousands of topologically interlocked DNA rings. Because of its highly unusual structure, kDNA has a complex replication mechanism. Our approach to understanding this mechanism is to identify the proteins involved and to characterize their function. So far approx. 30 candidate proteins have been discovered and we predict that there are over 100. To identify genes for more kDNA replication proteins, we are using an RNA interference library, which is the first forward genetic approach used for these parasites.

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

Replication of kDNA in the mitochondrion of the kinetoplastid protozoan is an essential process. One of the proteins that may be required for the kDNA replication is the ribonuclease H (RNase H; EC 3.1.26.4). We have identified four distinct ribonuclease H genes in Leishmania, one type I (LRNase HI) and three type II (LRNase HIIA, LRNase HIIB and LRNase HIIC). We detail here molecular characterization of LRNase HIIC. The coding sequence of LRNase HIIC is 1425 bp in length encoding a 474-amino acid protein with a calculated molecular mass of ∼53 kDa. While LRNase HIIC shares several conserved domains with mitochondrial RNase H from other organisms, it has three extra patches of amino acid sequences unique to this enzyme. Functional identity of this protein as an RNase H was verified by genetic complementation in RNase H-deficient Escherichia coli. The precursor protein may be enzymatically inactive as it failed to complement the E. coli mutant. The mitochondrial localization signal in LRNase HIIC is within the first 40 amino acid residues at the N-terminus. In vitro import of the protein by the mitochondrial vesicles showed that the precursor protein is processed to a 49-kDa protein. Antisense ablation of LRNase HIIC gene expression is lethal to the parasite cells both in vitro and in vivo. This study not only reveals the significance of the LRNase HIIC in the kinetoplast biology but also identifies a potential molecular target for antileishmanial chemotherapy.

The structure and replication of kinetoplast DNA.

Kinetoplast DNA (kDNA), the mitochondrial DNA of flagellated protozoa of the order Kinetoplastida, is unique in its structure, function and mode of replication. It consists of few dozen maxicircles, encoding typical mitochondrial proteins and ribosomal RNA, and several thousands minicircles, encoding guide RNA molecules that function in the editing of maxicircles mRNA transcripts. kDNA minicircles and maxicircles in the parasitic species of the family Trypanosomatidae are topologically linked, forming a two dimensional fishnet-type DNA catenane. Studies of early branching free-living and parasitic species of the Bodonidae family revealed various other forms of this remarkable DNA structure and suggested the evolution of kDNA from unlinked DNA circles and covalently-linked concatamers into a giant topological catenane. The replication of kDNA occurs during nuclear S phase and includes the duplication of free detached minicircles and catenated maxicircle and the generation of two progeny kDNA networks that segregate upon cell division. Recent reports of sequence elements and specific proteins that regulate the periodic expression of replication proteins advanced our understanding of the mechanisms that regulate the temporal link between mitochondrial and nuclear DNA synthesis in trypanosomatids. Studies on kDNA replication enzymes and binding proteins revealed their remarkable organization in clusters at defined sites flanking the kDNA disk, in correlation with the progress in the cell cycle and the process of kDNA replication. In this review I describe the recent advances in the study of kDNA and discuss some of the major challenges in deciphering the structure, replication and segregation of this remarkable DNA structure.

Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins.

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.

Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle.

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

The structure and replication of kinetoplast DNA.

The mitochondrial DNA of trypanosomatid protozoa, termed kinetoplast DNA (kDNA), is unique in its structure, function, and mode of replication. kDNA is a massive network, composed of thousands of topologically interlocked DNA circles, which resembles the chain mail of medieval armor. Each cell contains one network condensed into a disk-shaped structure within the matrix of its single mitochondrion. The kDNA circles are of two types, maxicircles present in a few dozen copies and minicircles present in several thousand copies. The maxicircles, which encode ribosomal RNAs and a few mitochondrial proteins, are similar in structure and genetic function to the mitochondrial DNA of other eukaryotes. Many maxicircle transcripts undergo editing, a remarkable process involving the insertion or deletion of uridine residues at specific sites. The minicircles encode small guide RNAs that control the specificity of editing. During kDNA replication, covalently closed minicircles are released from the network by a topoisomerase II. The free minicircles replicate as theta-structures within one of two complexes of replication proteins that are positioned on opposite sides of the kinetoplast disk. The progeny minicircles, which contain nicks or gaps, are attached to the network periphery. Maxicircles also replicate as theta-structures, but they remain linked to the network. As replication proceeds, the number of minicircles and maxicircles increases. When the network has doubled in size, all of the minicircle nicks and gaps are repaired, and the network splits in two. The two progeny networks then segregate into the daughter cells.

The configuration of DNA replication sites within the Trypanosoma brucei kinetoplast.

The kinetoplast is a concatenated network of circular DNA molecules found in the mitochondrion of many trypanosomes. This mass of DNA is replicated in a discrete "S" phase in the cell cycle. We have tracked the incorporation of the thymidine analogue 5-bromodeoxyuridine into newly replicated DNA by immunofluorescence and novel immunogold labeling procedures. This has allowed the detection of particular sites of replicated DNA in the replicating and segregating kinetoplast. These studies provide a new method for observing kinetoplast DNA (kDNA) replication patterns at high resolution. The techniques reveal that initially the pattern of replicated DNA is antipodal and can be detected both on isolated complexes and in replicating kDNA in vivo. In Trypanosoma brucei the opposing edges of replicating kDNA never extend around the complete circumference of the network, as seen in other kinetoplastids. Furthermore, crescent-shaped labeling patterns are formed which give way to labeling of most of the replicating kDNA except the characteristic midzone. The configuration of these sites of replicated DNA molecules is different to previous studies on organisms such as Crithidia fasciculata, suggesting differences in the timing of replication of mini and maxicircles and/or organization of the replicative apparatus in the kinetoplast of the African trypanosome.

Kinetoplast DNA maxicircles: networks within networks.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomes, is an enormous network of interlocked minicircles and maxicircles. We selectively removed minicircles from Trypanosoma equiperdum kDNA networks by restriction enzyme cleavage. Maxicircles remained in aggregates that were resistant to protease or RNase and contained no residual minicircles, but were resolved into circular monomers by topoisomerase II. Maxicircles thus form independent catenanes within kDNA networks. Heterogeneity in the size, composition, and organization of maxicircle catenanes reflects changes that occur during kDNA replication. The rosette-like arrangement of maxicircle catenanes is distinctly different from that of minicircle catenanes. Trypanosome kDNA networks reveal unique topological complexity: they are composed of entirely dissimilar catenanes that are in turn extensively interlocked with one another.

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.