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Paul T Englund - One of the best experts on this subject based on the ideXlab platform.
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network news the replication of Kinetoplast DNA
Annual Review of Microbiology, 2012Co-Authors: Robert E Jensen, Paul T EnglundAbstract:One of the most fascinating and unusual features of trypanosomatids, parasites that cause disease in many tropical countries, is their mitochondrial DNA. This genome, known as Kinetoplast DNA (kDNA), is organized as a single, massive DNA network formed of interlocked DNA rings. In this review, we discuss recent studies on kDNA structure and replication, emphasizing recent developments on replication enzymes, how the timing of kDNA synthesis is controlled during the cell cycle, and the machinery for segregating daughter networks after replication.
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a new function of trypanosoma brucei mitochondrial topoisomerase ii is to maintain Kinetoplast DNA network topology
Molecular Microbiology, 2008Co-Authors: Megan E Lindsay, Eva Gluenz, Keith Gull, Paul T EnglundAbstract:The mitochondrial genome of Trypanosoma brucei, called Kinetoplast DNA, is a network of topologically interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Kinetoplast DNA synthesis involves release of minicircles from the network, replication of the free minicircles and reattachment of the progeny. Here we report a new function of the mitochondrial topoisomerase II (TbTOP2mt). Although traditionally thought to reattach minicircle progeny to the network, here we show that it also mends holes in the network created by minicircle release. Network holes are not observed in wild-type cells, implying that this mending reaction is normally efficient. However, RNAi of TbTOP2mt causes holes to persist and enlarge, leading to network fragmentation. Remarkably, these network fragments remain associated within the mitochondrion, and many appear to be appropriately packed at the local level, even as the overall Kinetoplast organization is dramatically altered. The deficiency in mending holes is temporally the earliest observable defect in the complex TbTOP2mt RNAi phenotype.
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the rotational dynamics of Kinetoplast DNA replication
Molecular Microbiology, 2007Co-Authors: Yanan Liu, Paul T EnglundAbstract:Summary Kinetoplast DNA (kDNA), from trypanosomatid mitochondria, is a network containing several thousand catenated minicircles that is condensed into a diskshaped structure in vivo. kDNA synthesis involves release of individual minicircles from the network, replication of the free minicircles and reattachment of progeny at two sites on the network periphery ~180° apart. In Crithidia fasciculata, rotation of the kDNA disk relative to the antipodal attachment sites results in distribution of progeny minicircles in a ring around the network periphery. In contrast, Trypanosoma brucei progeny minicircles accumulate on opposite ends of the kDNA disk, a pattern that did not suggest Kinetoplast motion. Thus, there seemed to be two distinct replication mechanisms. Based on fluorescence microscopy of the kDNA network undergoing replication, we now report that the T. brucei Kinetoplast does move relative to the antipodal sites. Whereas the C. fasciculata Kinetoplast rotates, that from T. brucei oscillates. Kinetoplast motion of either type must facilitate orderly replication of this incredibly complex structure.
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role of p38 in replication of trypanosoma brucei Kinetoplast DNA
Molecular and Cellular Biology, 2006Co-Authors: Beiyu Liu, Henrik Molina, Dario E Kalume, Akhilesh Pandey, Jack D Griffith, Paul T EnglundAbstract: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 θ-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.
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overexpression of a cytochrome b5 reductase like protein causes Kinetoplast DNA loss in trypanosoma brucei
Journal of Biological Chemistry, 2006Co-Authors: Shawn A Motyka, Mark E Drew, Gokben Yildirir, Paul T EnglundAbstract:Abstract 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 b5 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 b5 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 b5 reductase-like protein could be partially rescued by simultaneously overexpressing tryparedoxin.
Joseph Shlomai - One of the best experts on this subject based on the ideXlab platform.
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direct monitoring of the stepwise condensation of Kinetoplast DNA networks
Scientific Reports, 2021Co-Authors: Nurit Yaffe, Dvir Rotem, Awakash Soni, Danny Porath, Joseph ShlomaiAbstract:Condensation and remodeling of nuclear genomes play an essential role in the regulation of gene expression and replication. Yet, our understanding of these processes and their regulatory role in other DNA-containing organelles, has been limited. This study focuses on the packaging of Kinetoplast DNA (kDNA), the mitochondrial genome of Kinetoplastids. Severe tropical diseases, affecting large human populations and livestock, are caused by pathogenic species of this group of protists. kDNA consists of several thousand DNA minicircles and several dozen DNA maxicircles that are linked topologically into a remarkable DNA network, which is condensed into a mitochondrial nucleoid. In vitro analyses implicated the replication protein UMSBP in the decondensation of kDNA, which enables the initiation of kDNA replication. Here, we monitored the condensation of kDNA, using fluorescence and atomic force microscopy. Analysis of condensation intermediates revealed that kDNA condensation proceeds via sequential hierarchical steps, where multiple interconnected local condensation foci are generated and further assemble into higher order condensation centers, leading to complete condensation of the network. This process is also affected by the maxicircles component of kDNA. The structure of condensing kDNA intermediates sheds light on the structural organization of the condensed kDNA network within the mitochondrial nucleoid.
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The Kinetoplast DNA of the Australian trypanosome, Trypanosoma copemani, shares features with Trypanosoma cruzi and Trypanosoma lewisi.
International Journal for Parasitology, 2018Co-Authors: Adriana Botero, Irit Kapeller, Joseph Shlomai, Crystal Cooper, Peta L. Clode, R.c. Andrew ThompsonAbstract:Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10 bp sequence) and CSB-2 (8 bp sequence) present lower interspecies homology, while CSB-3 (12 bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257 bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.
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enzymatic mechanism controls redox mediated protein DNA interactions at the replication origin of Kinetoplast DNA minicircles
Journal of Biological Chemistry, 2008Co-Authors: Dotan Sela, Nurit Yaffe, Joseph ShlomaiAbstract:Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.
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binding of the universal minicircle sequence binding protein at the Kinetoplast DNA replication origin
Journal of Biological Chemistry, 2006Co-Authors: Itay Onn, Irit Kapeller, Kawther Abuelneel, Joseph ShlomaiAbstract:Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to Kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.
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the structure and replication of Kinetoplast DNA
Current Molecular Medicine, 2004Co-Authors: Joseph ShlomaiAbstract:Abstract 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.
Dan S. Ray - One of the best experts on this subject based on the ideXlab platform.
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A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei
Molecular and cellular biology, 2010Co-Authors: Jane C. Hines, Dan S. RayAbstract:Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of Kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a Kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the Kinetoplast and is essential for both cell growth and Kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the Kinetoplast DNA. A 3'-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.
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structure of discontinuities in Kinetoplast DNA associated minicircles during s phase in crithidia fasciculata
Nucleic Acids Research, 2007Co-Authors: Jane C. Hines, Dan S. RayAbstract:Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20-30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3'OH and 5'deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180 degrees apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.
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Identification of new Kinetoplast DNA replication proteins in trypanosomatids based on predicted S-phase expression and mitochondrial targeting.
Eukaryotic cell, 2007Co-Authors: Yu Sun, Jane C. Hines, Dan S. RayAbstract:Trypanosomatid parasites contain an unusual form of mitochondrial DNA (Kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in Kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The Leishmania major protein P105 (LmP105) and Trypanosoma brucei protein P93 (TbP93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the LmP105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of TbP93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of TbP93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.
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the crithidia fasciculata kap1 gene encodes a highly basic protein associated with Kinetoplast DNA
Molecular and Biochemical Parasitology, 1998Co-Authors: Jane C. Hines, Dan S. RayAbstract:Abstract The Crithidia fasciculata KAP1 gene encodes a small basic protein (p21) associated with Kinetoplast DNA. The p21 protein has a nine amino acid cleavable presequence closely related to those of several other proteins targeted to the Kinetoplast and binds non-specifically to Kinetoplast minicircle DNA. The p21 protein also has a calculated p I of 13 with two amino acids (lysine and alanine) accounting for more than 50% of the residues and with 25 out of 28 lysine residues contained in the C-terminal half of the protein. Immunolocalization of p21 shows that the protein is found exclusively in the Kinetoplast with a localization distinctly different from the antipodal localization of Kinetoplast DNA topoisomerase and DNA polymerase. The KAP1 gene is a single copy gene and the KAP1 mRNA is present at a constant level throughout the cell cycle. This highly basic protein may play a role in the condensation or segregation of the Kinetoplast DNA.
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nucleus encoded histone h1 like proteins are associated with Kinetoplast DNA in the trypanosomatid crithidia fasciculata
Molecular and Cellular Biology, 1996Co-Authors: Jane C. Hines, Michele L Engel, D G Russell, Dan S. RayAbstract: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-His6-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.
Michele M. Klingbeil - One of the best experts on this subject based on the ideXlab platform.
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Estimating properties of Kinetoplast DNA by fragmentation reactions
Journal of Physics A: Mathematical and Theoretical, 2018Co-Authors: L Ibrahim, Pengyu Liu, Michele M. Klingbeil, Yuanan Diao, Javier ArsuagaAbstract:The mitochondrial DNA of trypanosomes, called Kinetoplast DNA (kDNA) contains thousands of minicircles that are topologically linked into a single structure that resembles a medieval chainmail. This biological chainmail is characterized by two parameters: the link type between minicircles, and the number of minicircles linked to each minicircle (i.e. the minicircle valence). In previous works, a protocol was proposed to determine the mean value of the minicircle valence. In these experiments, minicircles were excised from the network and the products compared with those obtained from fragmenting idealized structures. These idealized structures assumed a negligible variance in the distribution of valences of the initial network. It is therefore unclear to what extent this theoretical analysis captures the true topology of the kDNA network when kDNA samples are extracted from unsynchronized cells or from cells with silenced kDNA replication genes. Subsequent studies proposed that there is a critical percolation density during network formation. We asked whether this density can be estimated using fragmentation reactions. The goal of this work is to develop a mathematical method that can be used to estimate the mean valence of networks when the variance of the valence is non-negligible. We first show microscopy data on Crithidia fasciculata that, in agreement with the original experimental results, show a distribution of valences with nonzero variance. Second, we use computer simulations of network fragmentation to show that the predicted and actual mean valence are different when the valence distribution has nonzero variance. We propose a more general mathematical formulation and computer simulations of kDNA fragmentation to estimate this value. Last, we show that fragmentation experiments may lead to errors in the estimation of the critical percolation density since the collapsing density depends on the initial density of the network, and on the fragmentation reaction.
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orientation of DNA minicircles balances density and topological complexity in Kinetoplast DNA
PLOS ONE, 2015Co-Authors: Yuanan Diao, Michele M. Klingbeil, Victor Rodriguez, Javier ArsuagaAbstract:Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network.
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Three mitochondrial DNA polymerases are essential for Kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei.
Eukaryotic cell, 2011Co-Authors: David F. Bruhn, Mark P. Sammartino, Michele M. KlingbeilAbstract:Trypanosoma brucei, the causative agent of human African trypanosomiasis, has a complex life cycle that includes multiple life cycle stages and metabolic changes as the parasite switches between insect vector and mammalian host. The parasite's single mitochondrion contains a unique catenated mitochondrial DNA network called Kinetoplast DNA (kDNA) that is composed of minicircles and maxicircles. Long-standing uncertainty about the requirement of kDNA in bloodstream form (BF) T. brucei has recently eroded, with reports of posttranscriptional editing and subsequent translation of kDNA-encoded transcripts as essential processes for BF parasites. These studies suggest that kDNA and its faithful replication are indispensable for this life cycle stage. Here we demonstrate that three kDNA replication proteins (mitochondrial DNA polymerases IB, IC, and ID) are required for BF parasite viability. Silencing of each polymerase was lethal, resulting in kDNA loss, persistence of prereplication DNA monomers, and collapse of the mitochondrial membrane potential. These data demonstrate that kDNA replication is indeed crucial for BF T. brucei. The contributions of mitochondrial DNA polymerases IB, IC, and ID to BF parasite viability suggest that these and other kDNA replication proteins warrant further investigation as a new class of targets for the development of antitrypanosomal drugs.
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Stem-Loop Silencing Reveals that a Third Mitochondrial DNA Polymerase, POLID, Is Required for Kinetoplast DNA Replication in Trypanosomes
American Society for Microbiology, 2008Co-Authors: Julian Chandler, Anthula V. Vandoros, Brian Mozeleski, Michele M. KlingbeilAbstract:ABSTRACT Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei . Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol γ) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.
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closing the gaps in Kinetoplast DNA network replication
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Michele M. Klingbeil, Paul T EnglundAbstract:Trypanosomatids 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. Fig. 1. Electron micrograph of a segment of a kDNA network from C. fasciculata . Small loops are minicircles. [Reproduced with permission from ref. 1 (Copyright 2001, Elsevier Science).] In this issue of PNAS, Sinha et al. (4 …
Jane C. Hines - One of the best experts on this subject based on the ideXlab platform.
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A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei
Molecular and cellular biology, 2010Co-Authors: Jane C. Hines, Dan S. RayAbstract:Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of Kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a Kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the Kinetoplast and is essential for both cell growth and Kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the Kinetoplast DNA. A 3'-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.
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structure of discontinuities in Kinetoplast DNA associated minicircles during s phase in crithidia fasciculata
Nucleic Acids Research, 2007Co-Authors: Jane C. Hines, Dan S. RayAbstract:Kinetoplast DNA (kDNA) is a novel form of mitochondrial DNA consisting of thousands of interlocked minicircles and 20-30 maxicircles. The minicircles replicate free of the kDNA network but nicks and gaps in the newly synthesized strands remain at the time of reattachment to the kDNA network. We show here that the steady-state population of replicated, network-associated minicircles only becomes repaired to the point of having nicks with a 3'OH and 5'deoxyribonucleoside monophosphate during S phase. These nicks represent the origin/terminus of the strand and occur within the replication origins (oriA and oriB) located 180 degrees apart on the minicircle. Minicircles containing a new L strand have a single nick within either oriA or oriB but not in both origins in the same molecule. The discontinuously synthesized H strand contains single nicks within both oriA and oriB in the same molecule implying that discontinuities between the H-strand Okazaki fragments become repaired except for the fragments initiated within the two origins. Nicks in L and H strands at the origins persist throughout S phase and only become ligated as a prelude to network division. The failure to ligate these nicks until just prior to network division is not due to inappropriate termini for ligation.
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Identification of new Kinetoplast DNA replication proteins in trypanosomatids based on predicted S-phase expression and mitochondrial targeting.
Eukaryotic cell, 2007Co-Authors: Yu Sun, Jane C. Hines, Dan S. RayAbstract:Trypanosomatid parasites contain an unusual form of mitochondrial DNA (Kinetoplast DNA [kDNA]) consisting of a catenated network of several thousand minicircles and a smaller number of maxicircles. Many of the proteins involved in the replication and division of kDNA are likely to have no counterparts in other organisms and would not be identified by similarity to known replication proteins in other organisms. A new kDNA replication protein conserved in Kinetoplastids has been identified based on the presence of posttranscriptional regulatory sequences associated with S-phase gene expression and predicted mitochondrial targeting. The Leishmania major protein P105 (LmP105) and Trypanosoma brucei protein P93 (TbP93) localize to antipodal sites flanking the kDNA disk, where several other replication proteins and nascent minicircles have been localized. Like some of these kDNA replication proteins, the LmP105 protein is only present at the antipodal sites during S phase. RNA interference (RNAi) of TbP93 expression resulted in a cessation of cell growth and the loss of kDNA. Nicked/gapped forms of minicircles, the products of minicircle replication, were preferentially lost from the population of free minicircles during RNAi, suggesting involvement of TbP93 in minicircle replication. This approach should allow the identification of other novel proteins involved in the duplication of kDNA.
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the crithidia fasciculata kap1 gene encodes a highly basic protein associated with Kinetoplast DNA
Molecular and Biochemical Parasitology, 1998Co-Authors: Jane C. Hines, Dan S. RayAbstract:Abstract The Crithidia fasciculata KAP1 gene encodes a small basic protein (p21) associated with Kinetoplast DNA. The p21 protein has a nine amino acid cleavable presequence closely related to those of several other proteins targeted to the Kinetoplast and binds non-specifically to Kinetoplast minicircle DNA. The p21 protein also has a calculated p I of 13 with two amino acids (lysine and alanine) accounting for more than 50% of the residues and with 25 out of 28 lysine residues contained in the C-terminal half of the protein. Immunolocalization of p21 shows that the protein is found exclusively in the Kinetoplast with a localization distinctly different from the antipodal localization of Kinetoplast DNA topoisomerase and DNA polymerase. The KAP1 gene is a single copy gene and the KAP1 mRNA is present at a constant level throughout the cell cycle. This highly basic protein may play a role in the condensation or segregation of the Kinetoplast DNA.
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nucleus encoded histone h1 like proteins are associated with Kinetoplast DNA in the trypanosomatid crithidia fasciculata
Molecular and Cellular Biology, 1996Co-Authors: Jane C. Hines, Michele L Engel, D G Russell, Dan S. RayAbstract: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-His6-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.
