Article Figures & Data
Figures
- FIG. 1.
Conservation of DNA ligase motifs in mitochondrial DNA ligases. Six colinear sequence elements conserved in ATP-dependent DNA ligases are compared between trypanosomatid and vertebrate mitochondrial DNA ligases. The aligned sequences are from human ligase IIIα (Hu3α), mouse ligase IIIα (Mu3α), Xenopus ligase IIIα (Xl3α), T. brucei ligase kα (Tbkα), T. brucei ligase kβ (Tbkβ), L. major ligase kα (Lmkα), L. major ligase kβ (Lmkβ), C. fasciculata ligase kα (Cfkα), and C. fasciculata ligase kβ (Cfkβ). Conserved amino acid residues are indicated by shading.
- FIG. 2.
Localization of epitope-tagged DNA LIG kα. Cloned cells expressing HA-tagged DNA LIG kα were fixed and permeabilized on coverslips and then incubated with anti-HA monoclonal antibodies (12CA5) and rabbit polyclonal antibodies against the C. fasciculata kinetoplast topo II. The cells were then incubated with secondary antibodies (goat anti-mouse Alexa 488 and goat anti-rabbit 568). The coverslips were washed, incubated with DAPI, and then mounted with mounting medium. The images were captured on a Leica TCS-SP MP confocal and multiphoton inverted microscope. The LIG kα (green) and topo II (red) localizations are shown relative to those of the nuclear and kinetoplast DNAs (blue).
- FIG. 3.
Localization of epitope-tagged LIG kβ. Cloned cells expressing LIG kβ were immunostained and examined by confocal microscopy as described in the legend to Fig. 2.
- FIG. 4.
RNAi of T. brucei mitochondrial DNA ligases. (A) RNAi of LIG kβ; (B) RNAi of LIG kα. Cells were grown in the absence (filled diamond) or presence (open diamond) of tetracycline (1 μg/ml). The concentration of cells was determined each day, and cultures were diluted to maintain log phase. (Inset) Northern analysis of RNAi induction. Cells were grown in the absence (−) or presence (+) of tetracycline (Tet) for 48 h. The blot was also probed for α-tubulin genes as a loading control.
- FIG. 5.
Cell phenotype during LIG kα RNAi induction. Cells were induced with tetracycline, and on subsequent days, samples were removed, fixed, and stained with DAPI. Cells were scored by kDNA and nuclear phenotype. (A) Examples of phenotypes scored are as follows: anucleate, cells with no nucleus but with a kinetoplast; normal, cells with a nucleus and kinetoplast; no kDNA, cells with a nucleus but no kinetoplast; monster, cells with multiple nuclei and/or kinetoplasts. (B) Graph representing the observed frequency of each phenotype. The number of cells with a particular phenotype is expressed as a percentage of the total cells for that time point. Grey, anucleate; black, normal; white, no kDNA; diagonal stripes, monster.
- FIG. 6.
Southern analysis of free and network-associated minicircles during LIG kα RNAi induction. Total cellular DNA was isolated from cells at 0, 1, and 2 days after the addition of tetracycline (tet). Equal cell equivalents of the DNA with (+) or without (−) treatment with topoisomerase II were analyzed on a 1.5% agarose gel. After blotting the gel, the membrane was probed with a minicircle probe. Form I, covalently closed minicircles; form II, relaxed minicircles; form III, linear minicircles. Markers: lane 1, day 0 DNA treated with HindIII and XbaI; lane 2, purified kDNA treated with topoisomerase II.
- FIG. 7.
FACS analysis of LIG kα RNAi induced cells. Cells from 0, 3, and 5 days of induction were stained with dihydroethidium and analyzed on a BD LSR analytical flow cytometer. The resulting plot shows the relative fluorescence intensity versus the relative cell count.
- FIG. 8.
DNA ligase activity of LIG kα. Autoradiograph of reaction products electrophoresed on a denaturing acrylamide gel. 5′ end-labeled oligo(dT) was annealed to a poly(dA) backbone and incubated either alone (0) or with increasing amounts (1, 2, and 4 μl) of the purified recombinant LIG (rLIG) kα. Ligation reactions contained 1 mM ATP. vol., volume.
Tables
- TABLE 1.
Comparison of kinetoplastid mitochondrial DNA ligases
Ligase % Identity with protein sequence from: T. brucei LIG kα L. major LIG kα C. fasciculata LIG kα T. brucei LIG kβ L. major LIG kβ C. fasciculata LIG kβ T. brucei LIG kα 100 L. major LIG kα 41 100 C. fasciculata LIG kα 37 58 100 T. brucei LIG kβ 29 23 23 100 L. major LIG kβ 29 23 22 54 100 C. fasciculata LIG kβ 28 24 23 56 76 100 - TABLE 2.
Comparison of T. brucei DNA ligases with DNA ligases from higher eukaryotes
Ligase % Identity with protein sequence from: T. brucei LIG kα T. brucei LIG kβ T. brucei LIG I Homo sapiens LIG I Homo sapiens LIG III Homo sapiens LIG IV T. brucei LIG kα 100 T. brucei LIG kβ 30 100 T. brucei LIG I 8 8 100 Homo sapiens LIG I 6 6 35 100 Homo sapiens LIG III 7 8 18 18 100 Homo sapiens LIG IV 5 7 9 11 10 100
