The assembly of kinetoplast DNA

The unusual structure of the kinetoplast DNA (kDNA) of trypanosomatids requires unique replication mechanisms. Deciphering the mechanisms that regulate the network assembly has been a challenge for many years. A better understanding of these processes was facilitated by recent studies on the fine structure of resting and replicating kDNA networks. In this review, Joseph Shlomai discusses our current view of the structural and mechanistic aspects of the assembly of kinetoplast DNA.     

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

Eukaryotic DNA Replication

Abstract

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

Role of p38 in replication of Trypanosoma brucei kinetoplast DNA

Trypanosomes have an unusual mitochondrial genome, called kinetoplast DNA, that is a giant network containing thousands of interlocked minicircles. During kinetoplast DNA synthesis, minicircles are released from the network for replication as theta-structures, and then the free minicircle progeny reattach to the network. We report that a mitochondrial protein, which we term p38, functions in kinetoplast DNA replication. RNA interference (RNAi) of p38 resulted in loss of kinetoplast DNA and accumulation of a novel free minicircle species named fraction S. Fraction S minicircles are so underwound that on isolation they become highly negatively supertwisted and develop a region of Z-DNA. p38 binds to minicircle sequences within the replication origin. We conclude that cells with RNAi-induced loss of p38 cannot initiate minicircle replication, although they can extensively unwind free minicircles.     

Reversible decatenation of kinetoplast DNA by a DNA topoisomerase from trypanosomatids.

DNA topoisomerase activity detected in cell extracts of the trypanosomatid Crithidia fasciculata interlocks kinetoplast DNA duplex minicircles into huge catenane forms resembling the natural kinetoplast DNA networks found in trypanosomes. Catenation of duplex DNA circles is reversible and equilibrium is affected by ionic strength, and by spermidine. The reaction requires magnesium, is ATP dependent and is inhibited by high concentrations of novobiocin. Extensive homology between duplex DNA rings was not required for catenane formation since DNA circles with unrelated sequences could be interlocked into mixed network forms. Covalently sealed catenaned DNA circles are specifically used as substrates for decatenation. No such preference for covalently sealed duplex DNA rings was observed for catenate formation. Its catalytic properties and DNA substrate preference, suggest a potential role for this eukaryotic topoisomerase activity in the replication of kinetoplast DNA.     

Effect of acriflavin on the kinetoplast of Leishmania tarentolae. Mode of action and physiological correlates of the loss of kinetoplast DNA

The loss of kinetoplast DNA in Leishmania tarentolae, which occurs in the presence of low concentrations of acriflavin, was found to be a result of selective inhibition of replication of this DNA. Nuclear DNA synthesis was relatively unaffected and cell and kinetoplast division proceeded normally for several generations. An approximately equal distribution of parental kinetoplast DNA between daughter kinetoplasts resulted in a decrease in the average amount of DNA per kinetoplast. The final disappearance of the stainable kinetoplast DNA occurred at a cell division in which all the remaining visible kinetoplast DNA was retained by one of the daughter cells. The selective inhibition of kinetoplast DNA synthesis was caused by a selective localization of acriflavin in the kinetoplast. The apparent intracellular localization of dye and the extent of uptake at a low dye concentration could be manipulated, respectively, by varying the hemin (or protoporphyrin IX) concentration in the medium and by adding red blood cell extract (or hemoglobin). Hemin and protoporphyrin IX were found to form a complex with acriflavin. During growth in acriflavin, cells exhibited an increasing impairment of colony-forming ability and rate of respiration. No change in the electrophoretic pattern of total cell soluble proteins was apparent. The data fit the working hypothesis that the loss of kinetoplast DNA leads to a respiratory defect which then leads to a decrease in biosynthetic reactions and eventual cell death. A possible use of the selective localization of acriflavin in the kinetoplast to photooxidize selectively the kinetoplast DNA is suggested.     

A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization

Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.     

Fellowship of the rings: the replication of kinetoplast DNA

Kinetoplastid protozoa such as trypanosomes and Leishmania are important because they cause human disease. These parasites are named after one of their most unusual features, a mitochondrial DNA known as kinetoplast DNA (kDNA). Unlike all other DNA in nature, kDNA comprises a giant network of interlocked DNA rings with a topology resembling that of medieval chain mail. The replication of the kDNA network is more complex than previously thought, and the discovery of new proteins involved in this process is currently the best approach for illuminating the replication mechanism.     

Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani

Trypanosomatids are typified by uniquely configured mitochondrial DNA--the kinetoplast. The replication timing of kinetoplast DNA (kDNA) is closely linked to nuclear S phase, but nuclear and kinetoplast compartments display staggered timing of segregation, post-replication. Kinetoplast division is completed before nuclear division in Trypanosoma species while nuclear division is completed first in Crithidia species. Leishmania donovani is the causative agent of visceral leishmaniasis, a form of leishmanial infection that is often fatal. Cell cycle related studies in Leishmania are hampered by difficulties in synchronizing these cells. This report examines the replication/segregation pattern and morphology of the kinetoplast in L. donovani with the aim of determining if these traits can be used to assign cell cycle stage to individual cells. By labeling replicating cells with bromodeoxyuridine after synchronization with hydroxyurea, we find that although both nuclear and kDNA initiate replication in early S phase, nuclear division precedes kinetoplast segregation in 80% of the cells. The kinetoplast is roundish/short rod-like in G1 and in early to mid-S phase, but prominently elongated/bilobed in late S phase and early G2/M. These morphological traits and segregation pattern of the kinetoplast can be used as a marker for cell cycle stage in a population of asynchronously growing L. donovani promastigotes, in place of cell synchronization procedures or instead of using antibody staining for cell cycle stage marker proteins.     

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.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.     

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.     

The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis

The mitochondrial DNA of Trypanosoma brucei is organized in a complex structure called the kinetoplast. In this study, we define the complete kinetoplast duplication cycle in T. brucei based on three-dimensional reconstructions from serial-section electron micrographs. This structural model was enhanced by analyses of the replication process of DNA maxi- and minicircles. Novel insights were obtained about the earliest and latest stages of kinetoplast duplication. We show that kinetoplast S phase occurs concurrently with the repositioning of the new basal body from the anterior to the posterior side of the old flagellum. This emphasizes the role of basal body segregation in kinetoplast division and suggests a possible mechanism for driving the rotational movement of the kinetoplast during minicircle replication. Fluorescence in situ hybridization with minicircle- and maxicircle-specific probes showed that maxicircle DNA is stretched out between segregated minicircle networks, indicating that maxicircle segregation is a late event in the kinetoplast duplication cycle. This new view of the complexities of kinetoplast duplication emphasizes the dependencies between the dynamic remodelling of the cytoskeleton and the inheritance of the mitochondrial genome.     

Dynamic localization of Trypanosoma brucei mitochondrial DNA polymerase ID

Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication.     

Structural asymmetry and discrete nucleic acid subdomains in the Trypanosoma brucei kinetoplast

The mitochondrial genome of Trypanosoma brucei is contained in a specialized structure termed the kinetoplast. Kinetoplast DNA (kDNA) is organized into a concatenated network of mini and maxicircles, positioned at the base of the flagellum, to which it is physically attached. Here we have used electron microscope cytochemistry to determine structural and functional domains involved in replication and segregation of the kinetoplast. We identified two distinct subdomains within the kinetoflagellar zone (KFZ) and show that the unilateral filaments are composed of distinct inner and outer filaments. Ethanolic phosphotungstic acid (E-PTA) and EDTA regressive staining indicate that basic proteins and DNA are major constituents of the inner unilateral filaments adjoining the kDNA disc. This evidence for an intimate connection of the unilateral filaments in the KFZ with DNA provides support for models of minicircle replication involving vectorial export of free minicircles into the KFZ. Unexpectedly however, detection of DNA in the KFZ throughout the cell cycle suggests that other processes involving kDNA occur in this domain. We also describe a hitherto unrecognized, intramitochondrial, filamentous structure rich in basic proteins that links the kDNA discs during their segregation and is maintained between them for an extended period of the cell cycle.     

Overexpression of a cytochrome b5 reductase-like protein causes kinetoplast DNA loss in Trypanosoma brucei

The mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA), contains thousands of minicircles and dozens of maxicircles topologically interlocked in a network. To identify proteins involved in network replication, we screened an inducible RNA interference-based genomic library for cells that lose kinetoplast DNA. In one cloned cell line with inducible kinetoplast DNA loss, we found that the RNA interference vector had aberrantly integrated into the genome resulting in overexpression of genes down-stream of the integration site (Motyka, S. A., Zhao, Z., Gull, K., and Englund, P. T. (2004) Mol. Biochem. Parasitol. 134, 163-167). We now report that the relevant overexpressed gene encodes a mitochondrial cytochrome b(5) reductase-like protein. This overexpression caused kDNA loss by oxidation/inactivation of the universal minicircle sequence-binding protein, which normally binds the minicircle replication origin and triggers replication. The rapid loss of maxicircles suggests that the universal minicircle sequence-binding protein might also control maxicircle replication. Several lines of evidence indicate that the cytochrome b(5) reductase-like protein controls the oxidization status of the universal minicircle sequence-binding protein via tryparedoxin, a mitochondrial redox protein. For example, overexpression of mitochondrial tryparedoxin peroxidase, which utilizes tryparedoxin, also caused oxidation of the universal minicircle sequence-binding protein and kDNA loss. Furthermore, the growth defect caused by overexpression of cytochrome b(5) reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.     

Molecular machines in archaeal DNA replication

The archaeal DNA replication apparatus is a simplified version of that of eukaryotes and has attracted attention as a tractable model system for the orthologous, but significantly more complex eukaryal machinery. A variety of archaeal model organisms have been investigated with strong emphasis on structural and biochemical analyses of replication-associated proteins. In this review we will describe recent advances in understanding the properties of the replicative helicase, the MCM complex, and the role of the sliding clamp, PCNA, in mediating a range of protein-DNA transactions. Although both complexes form ring shaped assemblies, they play very distinct roles at the leading and trailing edges of the replication fork machinery respectively.