Trypanosoma brucei and related protists are distinguished from all other eukaryotes by an unusual mitochondrial genome known as kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Replication of this single nucleoid involves a release, replicate, and reattach mechanism for the thousands of catenated minicircles and requires at least three DNA polymerase (POLIB, POLIC and POLID) with similarity to E. coli DNA polymerase I. Like other proofreading replicative DNA polymerases, POLIB has both an annotated polymerase domain and an exonuclease domain. Predictive modelling of POLIB indicates that it has the canonical right hand polymerase structure with a unique and large 369 amino acid insertion within the polymerase domain (thumb region) homologous to E. coli RNase T. The goal of this study was to evaluate whether the polymerase domain is necessary for the essential replicative role of POLIB. To study the structure-function relationship, an RNAi-complementation system was designed to ectopically express POLIB variants in T. brucei that has endogenous POLIB silenced by RNAi.Control experiments expressing an ectopic copy of POLIB wildtype (IBWTPTP) or polymerase domain mutant (IBPol-PTP) in the absence of RNAi did not impact fitness in procyclic cells despite protein levels being 5 - 8.5 fold higher than endogenous POLIB levels. Immunofluorescence detection of the tagged variants indicated homogenous expression of the variants in a population of cells and negligible changes in kDNA morphology. Lastly, Southern blot analyses of cells expressing the IBWTPTP or IBPol-PTP variants indicated no changes in free minicircle species.
A dually inducible RNAi complementation system was designed and tested with the IBWTPTP and IBPol-PTP variants. Inductions of POLIB RNAi accompanied by ectopic expression of either variant using the standard 1 mg/ml tetracycline resulted in low protein levels of both variants while knockdown of the endogenous POLIB mRNA was greater than 85%. Increasing the tetracycline concentration to 4 mg/ml improved expression levels of both variants. However, levels of the ectopically expressed variants never exceeded that of endogenous POLIB. Using the 4 mg/ml induction conditions, complementation with IBWTPTP resulted in a partial rescue of the POLIB RNAi phenotype based on fitness curves, quantification of kDNA content and Southern blot analysis of free minicircles. IBWTPTP complementation resulted in gradual increase of IBWTPTP protein levels over the 10 day induction, and a small kDNA phenotype instead of the progressive loss of kDNA normally associated with POLIB RNAi. Additionally, the loss of free minicircles was delayed.
Complementation with the IBPol-PTP variant produced more consistent levels of IBPol-PTP protein although still below endogenous POLIB levels. Loss of fitness was similar to POLIB RNAi alone. However, a small kDNA phenotype emerged early after just four days of complementation and persisted for the remainder of the induction. The majority of the IBRNAi + IBPol-PTP population (70%) contained small kDNA compared to the parental POLIB RNAi or IBWTPTP complementation that had only 45% and 50% small kDNA, respectively. Lastly, the pattern of free minicircle loss closely resembled POLIB RNAi alone. Together, these data suggest that the dually inducible system results in a partial rescue with the IBWTPTP variant. Rescue with IBPol-PTP variant results in a noticeably different phenotype from either POLIB RNAi alone or IBWTPTP complementation indicating that the POLIB polymerase domain is likely essential for the in vivo role of POLIB during kDNA replication.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-2164 |
| Date | 20 October 2021 |
| Creators | Armstrong, Raveen |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
| Rights | http://creativecommons.org/licenses/by/4.0/ |
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