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Author ORCID Identifier

https://orcid.org/0000-0001-8847-4291

AccessType

Open Access Dissertation

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

Year Degree Awarded

2023

Month Degree Awarded

September

First Advisor

Michele Klingbeil

Subject Categories

Pathogenic Microbiology

Abstract

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.

DOI

https://doi.org/10.7275/35998237

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