Characterization of Tte UvrD Helicase Activity (2023)
Undergraduates: Ameyovi Kpodzo, Ben Nguyen, Sebastian Zagler, Ryan Dumais
Faculty Advisor: Thomas Freeman
Department: Chemistry
Helicases, a class of proteins that unwind DNA, are a crucial component of DNA mismatch repair (MMR).1 While the prokaryotic MMR process is well-understood, less is known about the eukaryotic MMR pathway. Recent work has shown that the MMR process in some thermophilic bacteria display great similarity to eukaryotic processes, making characterization of these prokaryotic thermophiles a promising avenue to bridge the gap in understanding between prokaryotic and eukaryotic MMR.4 In this investigation, Thermoanaerobacter tengcongensis (Tte) UvrD helicase was subjected to FRET and ATPase assays with slow-annealed and snap-frozen dsDNA to compare the rate and efficiency of DNA separation in the presence and absence of mismatch pairs. Native gel assays were also performed to quantify the amount of dsDNA unwound over time. Results from the FRET and native gel assays were largely unreliable due to abnormal efficiency values and insufficient dsDNA unwinding respectfully. However, the ATPase assays reveal that dsDNA unwinding follows pseudo-zeroth order kinetics with respect to UvrD concentration. Future studies optimizing FRET and native gel assay conditions must be performed to characterize the differential unwinding of snap-frozen and slow-annealed dsDNA.
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