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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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cell cycle dependent localization and properties of a second mitochondrial dna ligase in crithidia fasciculata
Eukaryotic Cell, 2006Co-Authors: Krishna Murari Sinha, Jane C. Hines, Dan S. RayAbstract:The mitochondrial DNA in Kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed Kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kβ) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kα). LIG kα localizes to the Kinetoplast primarily in cells that have completed mitosis and contain either a dividing Kinetoplast or two newly divided Kinetoplasts. Essentially all dividing or newly divided Kinetoplasts show localization of LIG kα. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kα to the kDNA but at a much lower frequency. The mRNA level of LIG kα varies during the cell cycle out of phase with that of LIG kβ. LIG kα transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kβ transcript levels are maximal during S phase. The LIG kα protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kα transcript and the instability of the LIG kα protein suggest a possible role of the ligase in regulating minicircle replication.
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disruption of the crithidia fasciculata kap1 gene results in structural rearrangement of the Kinetoplast disc
Molecular and Biochemical Parasitology, 2001Co-Authors: Julius Lukes, Jane C. Hines, Cory J Evans, Nuraly K Avliyakulov, Vidya P Prabhu, Junghuei Chen, Dan S. RayAbstract:The mitochondrial DNA (Kinetoplast DNA) in trypanosomatids exists as a highly organized nucleoprotein structure with the DNA consisting of thousands of interlocked circles. Four H1 histone-like proteins (KAP1, 2, 3 and 4) are associated with the Kinetoplast DNA in the trypanosomatid Crithidia fasciculata. We have disrupted both alleles of the KAP1 gene in this diploid protozoan and shown that expression of the KAP1 protein is eliminated. The mutant strain is viable but has substantial rearrangement of the Kinetoplast structure. Expression of the KAP1 protein from an episome restored expression of the KAP1 protein in the mutant strain and also restored a normal Kinetoplast structure. These studies provide evidence that the KAP1 protein is involved in Kinetoplast DNA organization in vivo but is nonessential for cell viability.
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the crithidia fasciculata rnh1 gene encodes both nuclear and mitochondrial isoforms of rnase h
Nucleic Acids Research, 2001Co-Authors: Michele L Engel, Jane C. Hines, Dan S. RayAbstract:The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in Kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the Kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the Kinetoplast. These results suggest that the Kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.
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the Kinetoplast structure specific endonuclease i is related to the 5 exo endonuclease domain of bacterial dna polymerase i and colocalizes with the Kinetoplast topoisomerase ii and dna polymerase β during replication
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Michele L Engel, Dan S. RayAbstract:The mitochondrial DNA (Kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated Kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5′ exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase β at antipodal sites flanking the Kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase β [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911–919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during Kinetoplast replication. Together, these results suggest a point of control for Kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.
Paul T. Englund - One of the best experts on this subject based on the ideXlab platform.
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the Kinetoplast duplication cycle in trypanosoma brucei is orchestrated by cytoskeleton mediated cell morphogenesis
Molecular and Cellular Biology, 2011Co-Authors: Eva Gluenz, Paul T. Englund, Megan L Povelones, Keith GullAbstract: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.
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tbpif1 a trypanosoma brucei mitochondrial dna helicase is essential for Kinetoplast minicircle replication
Journal of Biological Chemistry, 2010Co-Authors: Beiyu Liu, Gokben Yildirir, Jack D Griffith, Jianyang Wang, Gokhan Tolun, Paul T. EnglundAbstract:Kinetoplast DNA, the trypanosome mitochondrial genome, is a network of interlocked DNA rings including several thousand minicircles and a few dozen maxicircles. Minicircles replicate after release from the network, and their progeny reattach. Remarkably, trypanosomes have six mitochondrial DNA helicases related to yeast PIF1 helicase. Here we report that one of the six, TbPIF1, functions in minicircle replication. RNA interference (RNAi) of TbPIF1 causes a growth defect and Kinetoplast DNA loss. Minicircle replication intermediates decrease during RNAi, and there is an accumulation of multiply interlocked, covalently closed minicircle dimers (fraction U). In studying the significance of fraction U, we found that this species also accumulates during RNAi of mitochondrial topoisomerase II. These data indicate that one function of TbPIF1 is an involvement, together with topoisomerase II, in the segregation of minicircle progeny.
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the major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in african trypanosomes in vitro
Molecular and Biochemical Parasitology, 2000Co-Authors: Isabel Roditi, Terry W Pearson, Paul T. Englund, Stefan Ruepp, Robert P Beecroft, Susan C Welburn, Kuoyuan Hwa, Clive WellsAbstract:Abstract Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large ‘vacuole-like structure’ or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the Kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and Kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for Kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and Kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.
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A Mitochondrial DNA Primase from the Trypanosomatid Crithidia fasciculata
The Journal of biological chemistry, 1997Co-Authors: Paul T. EnglundAbstract:Abstract We have purified to near homogeneity a DNA primase from a mitochondrial fraction of the trypanosomatidCrithidia fasciculata. The enzyme is a single polypeptide chain of 28 kDa. Using a poly(dT) template and ATP as a substrate, the enzyme makes oligonucleotides of which the vast majority are about 10 nucleotides in size or smaller. With a single-stranded M13 DNA template and the four rNTPs as substrates, the enzyme makes heterogeneous oligonucleotides in the same size range. These oligonucleotides efficiently prime the synthesis of DNA by the Klenow DNA polymerase. Immunolocalization with antibodies against the purified enzyme confirms that the primase is mitochondrial. Furthermore, the enzyme localizes to specific regions of the cell’s single mitochondrion, above and below the condensed Kinetoplast DNA. The primase does not co-localize with the mitochondrial topoisomerase II and DNA polymerase β, both of which are associated with two protein complexes positioned on opposite sides of the Kinetoplast disc. These localization studies have significant implications for the mechanism of Kinetoplast DNA replication.
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Kinetoplast maxicircle dna replication in crithidia fasciculata and trypanosoma brucei
Molecular and Cellular Biology, 1995Co-Authors: Laura Rocco Carpenter, Paul T. EnglundAbstract:Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of Kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.
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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cell cycle dependent localization and properties of a second mitochondrial dna ligase in crithidia fasciculata
Eukaryotic Cell, 2006Co-Authors: Krishna Murari Sinha, Jane C. Hines, Dan S. RayAbstract:The mitochondrial DNA in Kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed Kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kβ) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kα). LIG kα localizes to the Kinetoplast primarily in cells that have completed mitosis and contain either a dividing Kinetoplast or two newly divided Kinetoplasts. Essentially all dividing or newly divided Kinetoplasts show localization of LIG kα. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kα to the kDNA but at a much lower frequency. The mRNA level of LIG kα varies during the cell cycle out of phase with that of LIG kβ. LIG kα transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kβ transcript levels are maximal during S phase. The LIG kα protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kα transcript and the instability of the LIG kα protein suggest a possible role of the ligase in regulating minicircle replication.
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disruption of the crithidia fasciculata kap1 gene results in structural rearrangement of the Kinetoplast disc
Molecular and Biochemical Parasitology, 2001Co-Authors: Julius Lukes, Jane C. Hines, Cory J Evans, Nuraly K Avliyakulov, Vidya P Prabhu, Junghuei Chen, Dan S. RayAbstract:The mitochondrial DNA (Kinetoplast DNA) in trypanosomatids exists as a highly organized nucleoprotein structure with the DNA consisting of thousands of interlocked circles. Four H1 histone-like proteins (KAP1, 2, 3 and 4) are associated with the Kinetoplast DNA in the trypanosomatid Crithidia fasciculata. We have disrupted both alleles of the KAP1 gene in this diploid protozoan and shown that expression of the KAP1 protein is eliminated. The mutant strain is viable but has substantial rearrangement of the Kinetoplast structure. Expression of the KAP1 protein from an episome restored expression of the KAP1 protein in the mutant strain and also restored a normal Kinetoplast structure. These studies provide evidence that the KAP1 protein is involved in Kinetoplast DNA organization in vivo but is nonessential for cell viability.
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the crithidia fasciculata rnh1 gene encodes both nuclear and mitochondrial isoforms of rnase h
Nucleic Acids Research, 2001Co-Authors: Michele L Engel, Jane C. Hines, Dan S. RayAbstract:The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in Kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the Kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the Kinetoplast. These results suggest that the Kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.
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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.
Keith Gull - One of the best experts on this subject based on the ideXlab platform.
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Detailed interrogation of trypanosome cell biology via differential organelle staining and automated image analysis
BMC Biology, 2012Co-Authors: Richard J Wheeler, Keith Gull, Eva GluenzAbstract:Background Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the Kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method. Results We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of Kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the Kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of Kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring Kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically. Conclusions Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana , Crithidia fasciculata and wild-type and mutant Trypanosoma brucei . Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.
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the Kinetoplast duplication cycle in trypanosoma brucei is orchestrated by cytoskeleton mediated cell morphogenesis
Molecular and Cellular Biology, 2011Co-Authors: Eva Gluenz, Paul T. Englund, Megan L Povelones, Keith GullAbstract: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.
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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 GullAbstract: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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structural asymmetry and discrete nucleic acid subdomains in the trypanosoma brucei Kinetoplast
Molecular Microbiology, 2007Co-Authors: Eva Gluenz, Michael K Shaw, Keith GullAbstract: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.
Michele L Engel - One of the best experts on this subject based on the ideXlab platform.
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the crithidia fasciculata rnh1 gene encodes both nuclear and mitochondrial isoforms of rnase h
Nucleic Acids Research, 2001Co-Authors: Michele L Engel, Jane C. Hines, Dan S. RayAbstract:The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in Kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the Kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the Kinetoplast. These results suggest that the Kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.
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the Kinetoplast structure specific endonuclease i is related to the 5 exo endonuclease domain of bacterial dna polymerase i and colocalizes with the Kinetoplast topoisomerase ii and dna polymerase β during replication
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Michele L Engel, Dan S. RayAbstract:The mitochondrial DNA (Kinetoplast DNA) of the trypanosomatid Crithidia fasciculata has an unusual structure composed of minicircles and maxicircles topologically interlocked into a single network and organized in a disc-shaped structure at the base of the flagellum. We previously purified a structure-specific endonuclease (SSE1), based on its RNase H activity, that is enriched in isolated Kinetoplasts. The endonuclease gene has now been cloned, sequenced, and found to be closely related to the 5′ exonuclease domain of bacterial DNA polymerase I proteins. Although the protein does not contain a typical mitochondrial leader sequence, the enzyme is shown to colocalize with a type II DNA topoisomerase and a DNA polymerase β at antipodal sites flanking the Kinetoplast disc. Cell synchronization studies with an epitope-tagged construct show that the localization of the endonuclease to the antipodal sites varies in a cell cycle-dependent manner similar to that of the DNA polymerase β [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911–919]. Immunofluorescent localization of SSE1 to the antipodal sites is only observed during Kinetoplast replication. Together, these results suggest a point of control for Kinetoplast DNA replication through the regulation of the availability of DNA replication proteins and a possible role for the antipodal sites in removal of RNA primers and the repair of gaps in newly replicated minicircles.
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a structure specific dna endonuclease is enriched in Kinetoplasts purified from crithidia fasciculata
Nucleic Acids Research, 1998Co-Authors: Michele L EngelAbstract: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.
