Biology subjects

Klingbeil, M. M.

Publications and source records attributed to Klingbeil, M. M..

3 recordsLinked to original sources

bioRxiv2025

A Trypanosome Trifecta: an independently tunable triple inducer system for genetic studies in Trypanosoma brucei.

Trypanosoma brucei is a model unicellular parasite for cellular and molecular genetic studies, but tools for more multiplexed experiments are limited. Tetracycline (Tet)-inducible gene regulation has been a long-standing and effective approach for overexpression, RNAi and genome wide screens. The single inducer Tet-On system has been foundational for studying essential genes that are required for biological processes and identifying potential drug targets. To achieve greater flexibility in experimental design, we capitalized upon previously described dual inducer systems that combined vanillic acid (Van) or cumate (Cym) with Tet as inducers. Here we report the development of a triple inducer system combining Cym, Van and Tet repressor elements to selectively regulate the expression of three genetic elements within a single cell line called PHITER. As proof of principle to demonstrate independent control, we adapted the previously characterized Van/Tet dual inducer RNAi complementation cell line, IBCompVaT for additional Cym inducible expression of an eGFP reporter. We provide evidence that each of the inducer elements operate independently with no evidence of leaky expression. In this system, Cym induction is specific and tunable. Additionally, simultaneous triple induction for POLIB RNAi complementation with wildtype POLIB and eGFP expression phenocopied the near complete rescue of POLIB RNAi previously reported. This system enables the study of complex and pleiotropic phenotypes through more sophisticated experimental design.

microbiology↗

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↗