TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance

Abstract

Summary The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.

A Passion for Parasites

“…The labeling was frequently found displaced from rather than at the network poles (93), although the harsh treatment may have altered the labeling pattern. When Jianyang Wang repeated metabolic labeling with 5-ethynyl-2Ј-deoxyuridine, which was detected by click chemistry under much gentler conditions (TBS containing 2 mM CuSO 4 ) with a fluorescent azide, only the poles were labeled (61). This finding argues against oscillation.…”

U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA

“…The localization of LIG kβ was previously reported as mainly nodal but not overlapping with that of Topo II, suggesting that these proteins may be contained in separate but associated complexes (Downey et al, 2004). LIG kβ also showed localization on both sides of the kDNA disk in a minority of cells, and the histone H1- like protein, Kap4, (Xu et al, 1996) and the mitochondrial helicase, TbPIF8 (Wang et al, 2012) both exhibited sheet-like configurations.…”

“…LIG kβ also shows localization on both sides of the kDNA disk in a minority of cells, as does the histone H1- like protein, Kap4 (Xu et al, 1996) and the helicase, TbPIF8 (Wang et al, 2012). Several DNA replication enzymes, PolIβ, PolIC and PIF2, are apparently localized in the kinetoflagellar mitochondrial matrix region opposite the distal side of the kDNA disk where replication of the decatenated closed minicircles occurs (Onn et al, 2006).…”

TbKAP6, a Mitochondrial HMG Box-Containing Protein in Trypanosoma brucei, Is the First Trypanosomatid Kinetoplast-Associated Protein Essential for Kinetoplast DNA Replication and Maintenance

“…The C-terminal 3ϫMyc tagging of one allele of TbKAP6 was conducted as described previously (20). The fixation, permeabilization, and immunostaining of TbKAP6-Myc cells were performed as described previously (21), using rabbit anti-Myc polyclonal antibody (1:200) (Santa Cruz) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200) (Molecular Probes).…”

“…DNA and RNA preparation, Southern, Northern, and Western blotting (28), kDNA isolation and DAPI staining (27), (29), electron microscopy of isolated kDNA (30) and thin sections (20), and TdT labeling (20) were performed as described.…”