scholarly journals Cell cycle localization dynamics of mitochondrial DNA polymerase IC in African trypanosomes

2018 ◽  
Vol 29 (21) ◽  
pp. 2540-2552 ◽  
Author(s):  
Jeniffer Concepción-Acevedo ◽  
Jonathan C. Miller ◽  
Michael J. Boucher ◽  
Michele M. Klingbeil
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dynamic Environment ◽  
African Trypanosomes ◽  
Protein Levels ◽  
Replication Sites ◽  
Cycle Dependent ◽  
Mitochondrial Dna Polymerase

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.

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

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.

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

2010 ◽  
Vol 75 (6) ◽  
pp. 1414-1425 ◽  
Author(s):  
David F. Bruhn ◽  
Brian Mozeleski ◽  
Laurie Falkin ◽  
Michele M. Klingbeil
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Dna Polymerase ◽  
African Trypanosomes ◽  
Mitochondrial Dna Polymerase ◽  
Minicircle Dna

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

2020 ◽  
Vol 133 (9) ◽  
pp. jcs233072 ◽  
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

scholarly journals Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption

2012 ◽  
Vol 302 (9) ◽  
pp. G914-G924 ◽  
Author(s):  
Raymond G. Fox ◽  
Scott Magness ◽  
Gregory C. Kujoth ◽  
Tomas A. Prolla ◽  
Nobuyo Maeda
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Small Intestine ◽  
Dna Polymerase ◽  
Progenitor Cell ◽  
Accelerated Aging ◽  
Fold Increase ◽  
Dietary Lipids ◽  
Mitochondrial Dna Polymerase ◽  
The Impact

Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg D257A, that exhibit accelerated aging phenotype. As early as 3–7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

scholarly journals Changes in Organization of Crithidia fasciculata Kinetoplast DNA Replication Proteins during the Cell Cycle

1998 ◽  
Vol 143 (4) ◽  
pp. 911-919 ◽  
Author(s):  
Catharine E. Johnson ◽  
Paul T. Englund
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Topoisomerase Ii ◽  
Replication Proteins ◽  
Kinetoplast Dna ◽  
Dna Polymerase Β ◽  
Cycle Dependent

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle–dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein–dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase β colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase β is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

scholarly journals 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 ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1809-1824 ◽  
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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