Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.
Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.
Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.
Author ORCID Identifier
Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
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
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.
Delzell, Stephanie B., "A Biochemical Approach to Characterize a Divergent Trypanosoma brucei Mitochondrial DNA Polymerase, POLIB" (2023). Doctoral Dissertations. 2978.