Biology subjects

Delzell, S. B.

Publications and source records attributed to Delzell, S. B..

3 recordsLinked to original sources

bioRxiv2025

An independently tunable dual control system for RNAi complementation in Trypanosoma brucei

Trypanosoma brucei is a tractable protist parasite for which many genetic tools have been developed to study novel biology. A striking feature of T. brucei is the catenated mitochondrial DNA network called the kinetoplast DNA (kDNA) that is essential for parasite survival and life cycle completion. Maintenance of kDNA requires three independently essential paralogs that have homology to bacterial DNA polymerase I (POLIB, POLIC and POLID). We previously demonstrated that POLIB has a divergent domain architecture that displayed enzymatic properties atypical for replicative DNA polymerases. To evaluate the functional domains required for kDNA replication in vivo, we pursued an RNAi complementation approach based on the widely used tetracycline (Tet) single inducer system. Tet induction of RNAi and complementation with wildtype POLIB (POLIBWT) resulted in a 93% knockdown of endogenous POLIB mRNA but insufficient ectopic POLIBWT expression. This incomplete rescue emphasized the need for a more versatile induction system that will allow independent, tunable, and temporal regulation of gene expression. Hence, we adapted a dual control vanillic acid (Van)-Tet system that can independently control gene expression for robust RNAi complementation. Dual induction with Van and Tet (RNAi + Overexpression) resulted in 91% endogenous POLIB knockdown accompanied by robust and sustained ectopic expression of POLIBWT, and a near complete rescue of the POLIB RNAi defects. To more precisely quantify changes in kDNA size during RNAi, we also developed a semi-automated 3D image analysis tool to measure kDNA volume. Here we provide proof of principle for a dual inducer system that allows more flexible control of gene expression to perform RNAi and overexpression independently or concurrently within a single cell line. This system overcomes limitations of the single inducer system and can be valuable for elegant mechanistic studies in the field.

molecular biology↗

bioRxiv2022

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb 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 fulfill the role of a replicative, proofreading enzyme remains enigmatic. De novo modeling of POLIB revealed a structure 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 in a non-processive manner. The 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 is able to extend 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 corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The unique POLIB thumb insertion influences the balance between polymerase and exonuclease activity and provides another example of exquisite diversity among DNA polymerases for specialized function.

biochemistry↗

bioRxiv2019

Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes.

The 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 the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.\n\nSummary statementMitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase.

microbiology↗