Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

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

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 a single nucleoid requires at least three DNA polymerase I-like proteins (i.e. POLIB, POLIC and POLID), each showing discrete localizations near the kDNA during S phase. POLIB and POLID have roles in minicircle replication but the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNA interference (RNAi)-complementation system to dissect the functions of two distinct POLIC regions, i.e. the conserved family A DNA polymerase (POLA) domain and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization of kDNA, active site point mutations in the POLA domain impaired minicircle replication similar to that of POLIB and POLID depletions. Complementation with POLA domain alone abolished the formation of POLIC foci and partially rescued the RNAi phenotype. Furthermore, we provide evidence that the UCR is crucial in cell cycle-dependent protein localization and facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase that impacts on mitochondrial nucleoid distribution.This article has an associated First Person interview with the first author of the paper.