Protein Purification and Analysis of a Human DNA Polymerase Theta Helicase Mutant and Trypanosome Orthologue
Mackenzie, Kenneth
Mackenzie, Kenneth
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Abstract
DNA Polymerase Theta (Polθ), the enzyme responsible for the microhomologous-mediated end-joining (MMEJ) repair pathway of double stranded DNA breaks, has been identified as a drug target due to its role in treatment resistant cancers. The enzyme is comprised of a C-terminal polymerase domain, a central domain of unknown function, and an N-terminal helicase-like domain. Polθ has been marked for study due to the unknown nature of the helicase-like domain whose role in MMEJ has eluded scientists due to its lack of helicase activity. In this study, the Trypanosoma cruzi orthologue as well as the human variant KM16 of human Polθ were produced and purified for attempted structural studies. Using Ni-NTA nickel affinity, heparin, Q FF, histrap, and gel filtration chromatography columns, we attempted to isolate the protein and use dynamic light scattering to assess the structural radius of the orthologue and variant constructs. In the end both proteins evaded purification, but much progress was made on the development of a reliable protocol for isolating each protein.
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2023-01-01