Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata

Abstract

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

FIG. 1.

Immunolocalization of HA-tagged LIG kα. (A) A dividing cell stained with mouse HA.11 antibodies conjugated with Alexa Fluor 488 (a) and DAPI (b) and a phase-contrast image (c) are shown. A cell with a single nucleus stained overnight with mouse HA.11 antibodies conjugated with Alexa Fluor 488 (d) and with DAPI (e) and a phase-contrast image (f) are also shown. (B) Dividing cells stained with mouse 12CA5 antibodies and goat anti-mouse IgG conjugated with Alexa Fluor 594 and with rabbit anti-RPA1 and goat anti-rabbit IgG conjugated with Alexa Fluor 488 to stain the nucleus (a and e) and DAPI (b), a DAPI and AlexaFluor488 merged image (f), phase images (c and g), and merged images (d and h) are shown. (C) Cells with a single nucleus stained with mouse 12CA5 antibodies and goat anti-mouse IgG conjugated with AlexaFluor 594 and with DAPI (a) and a phase image (b). (D) Confocal image of a dividing cell stained with mouse HA.11 antibodies conjugated with Alexa Fluor 488 and with DAPI shown in false color (red) (a); a phase image is also shown (b).

FIG. 2.

Purification of recombinant LIG kα. (A) Metal chelate chromatography of His-tagged LIG kα. Lane 1, molecular mass markers (kDa); lane 2, total cell extract; lane 3, column load; lane 4, flow through; lane 5, wash with loading buffer; lane 6, wash with 60 mM imidazole in loading buffer; lanes 7 to 13, elution with 0.3 M imidazole in column buffer. (B) G25 Sephadex desalting column of fraction 9 from panel A. Lane 1, molecular mass markers (in kilodaltons); lane 2, column load; lanes 3 to 13, eluted fractions.

FIG. 3.

Adenylation and ligation by the recombinant LIG kα. (A) Lane 1, adenylation of 20 ng of the recombinant protein in the presence of [α-³²P]ATP. Deadenylation of the adenylated protein upon incubation with 5 μg of DNase I-treated calf thymus DNA (lane 3), 2.5 μg of poly(dA) · oligo(dT) (lane 4), or 2.5 μg of poly(rA) · oligo(dT) (lane 5). Lane 2 shows a mock deadenylation reaction. Reaction products were analyzed by sodium dodecyl sulfate-gel electrophoresis and autoradiography of the dried gel. (B) DNA ligation by recombinant LIG kα. 5′ End-labeled oligo(dT)₂₀ annealed to poly(dA) was incubated with 0, 5, or 10 ng of the recombinant protein (lanes 1 to 3, respectively) in the presence of 1 mM ATP. The reaction products were analyzed on a urea-12% polyacrylamide gel and imaged by autoradiography of the dried gel.

FIG. 4.

Transcript levels of LIG kα and LIG kβ genes during the cell cycle. (A) Transcript levels of LIG kα, LIG kβ, and DHFR-TS genes in a synchronized C. fasciculata culture were analyzed by Northern blot after release from a hydroxyurea block. Numbers at the top indicate the time at which cell aliquots were collected for RNA isolation after hydroxyurea release. (B) PhosphorImager quantitation of the transcript levels in panel A. The relative transcript levels are shown as a function of time after release from the hydroxyurea block.

FIG. 5.

Sequences related to mRNA cycling sequence elements flanking kinetoplast DNA ligase genes. The highly conserved central hexamer ATAGAA of the consensus octamer cycling sequence (7, 34) and the related sequence ATAGA are shown flanking the coding sequences of LIG kα and LIG kβ.

FIG. 6.

Turnover of LIG kα in the absence of protein synthesis. C. fasciculata cultures expressing HA epitope-tagged LIG kα (A) and HA epitope-tagged LIG kβ (B) were treated with cycloheximide at 100 μg/ml. Cell aliquots were collected at 1-h intervals and analyzed by Western blotting of total cell extracts by probing with 12CA5 monoclonal antibodies and rabbit polyclonal antibodies to the CSBPA protein used as loading control.