Stem-loop silencing reveals that a third mitochondrial DNA polymerase, POLID, is required for kinetoplast DNA replication in trypanosomes

Abstract

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomes, is a catenated network containing thousands of minicircles and tens of maxicircles. The topological complexity dictates some unusual features including a topoisomerase-mediated release-and-reattachment mechanism for minicircle replication and at least six mitochondrial DNA polymerases (Pols) for kDNA transactions. Previously, we identified four family A DNA Pols from Trypanosoma brucei with similarity to bacterial DNA Pol I and demonstrated that two (POLIB and POLIC) were essential for maintaining the kDNA network, while POLIA was not. Here, we used RNA interference to investigate the function of POLID in procyclic T. brucei. Stem-loop silencing of POLID resulted in growth arrest and the progressive loss of the kDNA network. Additional defects in kDNA replication included a rapid decline in minicircle and maxicircle abundance and a transient accumulation of minicircle replication intermediates before loss of the kDNA network. These results demonstrate that POLID is a third essential DNA Pol required for kDNA replication. While other eukaryotes utilize a single DNA Pol (Pol gamma) for replication of mitochondrial DNA, T. brucei requires at least three to maintain the complex kDNA network.

FIG. 1.

Effects of POLID RNAi. (A) Diagram: protein domain structure of POLID with the region used to generate the pSLID and pZJMID2 vectors (1) and the region used to generate pZJMID1 (2) (21). Graph: clonal cell line C8P1 was grown in the absence (closed circles) or presence (open diamonds) of tetracycline (1 μg/ml) to express the stem-loop dsRNA. (B) Northern blot of total RNA from uninduced (−) and RNAi-induced (+) cultures hybridized for 48 h with radiolabeled probes specific for POLID, POLIC, and POLIB. Hybridization with α-tubulin probe was the loading control. RNA marker sizes are indicated on the left in kilobases. (C) Kinetics of kDNA loss as determined by visual analysis. Symbols: filled circles, normally sized kDNA; open squares, small kDNA; open triangles, no kDNA. More than 300 randomly selected cells were scored for each time point. (D) Effect of POLID RNAi on kinetoplast size. (Top) DIC images; (bottom) DAPI-stained fluorescent images. Abbreviations: N, nucleus; K, normally sized kDNA; sK, small kDNA; no K, no kDNA; Un, uninduced. Bar, 10 μm.

FIG. 2.

POLID RNAi effects on kDNA abundance. C8P1 clonal cells were grown in the presence or absence of tetracycline and harvested at the indicated time points. (A) Total DNA (10⁶ cell equivalents/sample) was digested, fractionated on a 1% agarose gel, and transferred to a nylon membrane. Southern blot analysis of changes in minicircle (closed circles) and maxicircle (open squares) content was determined with minicircle, maxicircle, and tubulin probes. Quantitation of kDNA species was performed using a PhosphorImager, and values are reported as percentages of those detected for uninduced cells. Values are the mean (± standard deviation) of three separate RNAi inductions. (B) Southern analysis of free minicircles. Total DNA (2 × 10⁶ cells/lane) was fractionated on a 1.5% agarose gel and transferred to a nylon membrane. After a probe for minicircle DNA, the blot was stripped and probed for α-tubulin as the loading control. *, multiply gapped minicircle progeny; CM, catenated minicircles. (C) Quantitation of free minicircles by phosphorimaging. Values were corrected for the loading control. Symbols: closed circles, CC free minicircles; open squares, N/G free minicircles. Values are the mean (± standard deviation) of two separate RNAi inductions, one of which is shown in panel B.