Trypanosoma brucei is a single-celled parasitic protist that causes African sleeping sickness in people and nagana in cattle in sub-Saharan Africa. T. brucei and related trypanosomatid parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of dozens of 23 kb maxicircles and thousands of 1 kb minicircles. The kDNA structure and replication mechanism are divergent from other eukaryotes and essential for parasite survival. POLIB is one of three Family A DNA polymerases that are independently essential to maintain the kDNA network, and has been implicated in minicircle replication. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme remains enigmatic. De novo modelling 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. In chapter 2, we explore the polymerase and exonuclease activity of POLIB using purified his-tagged v recombinant variants that have been truncated and codon optimized for expression in E. coli. Using this recombinant protein variants we demonstrated that the 3'-5' exonuclease activity of recombinant POLIB prefers DNA vs. RNA substrates and prefers singlestranded vs. double-stranded substrates. POLIB 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 RNA or DNA primers. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. We also show that overexpression of the exonuclease-ablated variant of POLIB in T. brucei results in a loss of fitness and impacts kDNA replication. We postulate that this unique enzyme and the machinery associated with it in the process of kDNA replication could be excellent drug targets worthy of further study.