Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

ABSTRACT 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.

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Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.05008-11 ◽

2011 ◽

Vol 10 (6) ◽

pp. 734-743 ◽

Cited By ~ 18

Author(s):

David F. Bruhn ◽

Mark P. Sammartino ◽

Michele M. Klingbeil

Keyword(s):

Mitochondrial Dna ◽

Life Cycle ◽

Trypanosoma Brucei ◽

Dna Polymerases ◽

Bloodstream Form ◽

Complex Life Cycle ◽

Replication Proteins ◽

Kinetoplast Dna ◽

Content Type ◽

Parasite Viability

ABSTRACT 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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A Second Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast Minicircle DNA Replication in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00308-10 ◽

2011 ◽

Vol 10 (3) ◽

pp. 445-454 ◽

Cited By ~ 24

Author(s):

Jane C. Hines ◽

Dan S. Ray

Keyword(s):

Mitochondrial Dna ◽

Trypanosoma Brucei ◽

Rna Recognition Motif ◽

Dependent Manner ◽

Dna Viruses ◽

Content Type ◽

Amino Terminal ◽

Circular Dnas ◽

Nucleocytoplasmic Large Dna Viruses ◽

Minicircle Dna

ABSTRACT The mitochondrial DNA of trypanosomes contains two types of circular DNAs, minicircles and maxicircles. Both minicircles and maxicircles replicate from specific replication origins by unidirectional theta-type intermediates. Initiation of the minicircle leading strand and also that of at least the first Okazaki fragment involve RNA priming. The Trypanosoma brucei genome encodes two mitochondrial DNA primases, PRI1 and PRI2, related to the primases of eukaryotic nucleocytoplasmic large DNA viruses. These primases are members of the archeoeukaryotic primase superfamily, and each of them contain an RNA recognition motif and a PriCT-2 motif. In Leishmania species, PRI2 proteins are approximately 61 to 66 kDa in size, whereas in Trypanosoma species, PRI2 proteins have additional long amino-terminal extensions. RNA interference (RNAi) of T. brucei PRI2 resulted in the loss of kinetoplast DNA and accumulation of covalently closed free minicircles. Recombinant PRI2 lacking this extension (PRI2ΔNT) primes poly(dA) synthesis on a poly(dT) template in an ATP-dependent manner. Mutation of two conserved aspartate residues (PRI2ΔNTCS) resulted in loss of enzymatic activity but not loss of DNA binding. We propose that PRI2 is directly involved in initiating kinetoplast minicircle replication.

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The Mitochondrial Ca2+Uniporter Complex (MCUC) ofTrypanosoma bruceiIs a Hetero-oligomer That Contains Novel Subunits Essential for Ca2+Uptake

mBio ◽

10.1128/mbio.01700-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 16

Author(s):

Guozhong Huang ◽

Roberto Docampo

Keyword(s):

Trypanosoma Brucei ◽

Mitochondrial Calcium ◽

Transmembrane Helices ◽

Yeast Two Hybrid ◽

Content Type ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Essential Components ◽

Split Ubiquitin ◽

Two Hybrid

ABSTRACTThe mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterizedTrypanosoma brucei’s MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralogT. bruceiMCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids.In situtagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria ofT. brucei. Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca2+uptake inT. brucei, respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC ofT. bruceiand defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC.IMPORTANCETrypanosoma bruceicauses human African trypanosomiasis and nagana in animals. The finding of a mitochondrial calcium uniporter (MCU) conserved in this parasite was essential for the discovery of the gene encoding the pore subunit. Mitochondrial Ca2+transport mediated by the MUC complex is critical inTrypanosoma bruceifor shaping the dynamics of cytosolic Ca2+increases, for the bioenergetics of the cells, and for viability and infectivity. We found that one component of the complex (MCUb) does not act as a dominant negative effector of the channel as in vertebrate cells and that the TbMCUC possesses two unique subunits (MCUc and MCUd) present only in trypanosomatids and required for Ca2+transport. The study of the interactions between these four subunits (MCU, MCUb, MCUc, and MCUd) by a variety of techniques that include coimmunoprecipitation, split-ubiquitin membrane-based yeast two-hybrid assays, and site-directed mutagenesis suggests that they interact through their transmembrane helices to form hetero-oligomers.

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Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421733112 ◽

2015 ◽

Vol 112 (28) ◽

pp. 8596-8601 ◽

Cited By ~ 16

Author(s):

Christal D. Sohl ◽

Michal R. Szymanski ◽

Andrea C. Mislak ◽

Christie K. Shumate ◽

Sheida Amiralaei ◽

...

Keyword(s):

Mitochondrial Dna ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Biologically Active ◽

Striking Difference ◽

Substrate Selection ◽

Human Mitochondrial Dna ◽

Lower Toxicity ◽

Essential Components ◽

Mitochondrial Dna Polymerase

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

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Faculty Opinions recommendation of Premature ageing in mice expressing defective mitochondrial DNA polymerase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019285.219437 ◽

2004 ◽

Author(s):

David Rand

Keyword(s):

Mitochondrial Dna ◽

Dna Polymerase ◽

Premature Ageing ◽

Mitochondrial Dna Polymerase

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The tamas Gene, Identified as a Mutation That Disrupts Larval Behavior in Drosophila melanogaster, Codes for the Mitochondrial DNA Polymerase Catalytic Subunit (DNApol-γ125)

Genetics ◽

10.1093/genetics/153.4.1809 ◽

1999 ◽

Vol 153 (4) ◽

pp. 1809-1824 ◽

Cited By ~ 8

Author(s):

Balaji Iyengar ◽

John Roote ◽

Ana Regina Campos

Keyword(s):

Mitochondrial Dna ◽

Drosophila Melanogaster ◽

Mutant Strain ◽

Dna Polymerase ◽

Complementation Group ◽

Behavioral Changes ◽

Catalytic Subunit ◽

Primary Deficit ◽

Mitochondrial Dna Polymerase ◽

Food Substrate

AbstractFrom a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as “dark inertia”). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-γ125).

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