Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes

Trypanosoma brucei has a unique catenated mitochondrial DNA (mtDNA) network called kinetoplast DNA (kDNA). Replication of kDNA occurs once per cell cycle in near synchrony with nuclear S phase and requires the coordination of many proteins. Among these are three essential DNA polymerases (TbPOLIB, IC, and ID). Localization dynamics of these proteins with respect to kDNA replication stages and how they coordinate their functions during replication are not well understood. We previously demonstrated that TbPOLID undergoes dynamic localization changes that are coupled to kDNA replication events. Here, we report the localization of TbPOLIC, a second essential DNA polymerase, and demonstrate the accumulation of TbPOLIC foci at active kDNA replication sites (antipodal sites) during stage II of the kDNA duplication cycle. While TbPOLIC was undetectable by immunofluorescence during other cell cycle stages, steady-state protein levels measured by Western blot remained constant. TbPOLIC foci colocalized with the fraction of TbPOLID that localized to the antipodal sites. However, the partial colocalization of the two essential DNA polymerases suggests a highly dynamic environment at the antipodal sites to coordinate the trafficking of replication proteins during kDNA synthesis. These data indicate that cell cycle–dependent localization is a major regulatory mechanism for essential mtDNA polymerases during kDNA replication.

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A DNA polymerization-independent role for mitochondrial DNA polymerase I-like protein C in African trypanosomes

Journal of Cell Science ◽

10.1242/jcs.233072 ◽

2020 ◽

Vol 133 (9) ◽

pp. jcs233072 ◽

Cited By ~ 1

Author(s):

Jonathan C. Miller ◽

Stephanie B. Delzell ◽

Jeniffer Concepción-Acevedo ◽

Michael J. Boucher ◽

Michele M. Klingbeil

Keyword(s):

Mitochondrial Dna ◽

Dna Polymerase ◽

Protein C ◽

Dna Polymerase I ◽

African Trypanosomes ◽

Dna Polymerization ◽

Polymerase I ◽

Mitochondrial Dna Polymerase

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

Molecular and Cellular Biology ◽

10.1128/mcb.01231-09 ◽

2010 ◽

Vol 30 (6) ◽

pp. 1319-1328 ◽

Cited By ~ 24

Author(s):

Jane C. Hines ◽

Dan S. Ray

Keyword(s):

Mitochondrial Dna ◽

Dna Replication ◽

Cell Growth ◽

Trypanosoma Brucei ◽

Dna Polymerase ◽

Rnase H ◽

Kinetoplast Dna ◽

African Trypanosomes ◽

Dna Primase

ABSTRACT 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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Disease Mutations in the Human Mitochondrial DNA Polymerase Thumb Subdomain Impart Severe Defects in Mitochondrial DNA Replication

Journal of Biological Chemistry ◽

10.1074/jbc.m109.011940 ◽

2009 ◽

Vol 284 (29) ◽

pp. 19501-19510 ◽

Cited By ~ 30

Author(s):

Rajesh Kasiviswanathan ◽

Matthew J. Longley ◽

Sherine S. L. Chan ◽

William C. Copeland

Keyword(s):

Mitochondrial Dna ◽

Dna Replication ◽

Dna Polymerase ◽

Human Mitochondrial Dna ◽

Disease Mutations ◽

Mitochondrial Dna Replication ◽

Mitochondrial Dna Polymerase

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Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes

10.1101/730523 ◽

2019 ◽

Author(s):

Jonathan C Miller ◽

Stephanie B Delzell ◽

Jeniffer Concepción-Acevedo ◽

Michael J Boucher ◽

Michele M Klingbeil

Keyword(s):

Cell Cycle ◽

Mitochondrial Dna ◽

Dna Polymerase ◽

Function Analysis ◽

Point Mutations ◽

Rnai Phenotype ◽

African Trypanosomes ◽

Dna Segregation ◽

Summary Statement ◽

Mitochondrial Dna Polymerase

ABSTRACTThe mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA). Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.Summary statementMitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase.

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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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