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bioRxiv · 10.1101/2021.01.05.425498

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Carney, S. P.·Ma, W.·Whitley, K. D.·Jia, H.·Lohman, T. M.·Luthey-Schulten, Z. A.·Chemla, Y. R.
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Abstract

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

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Carney, S. P., Ma, W., Whitley, K. D., Jia, H., Lohman, T. M., Luthey-Schulten, Z. A., Chemla, Y. R.. 2021-01-06. Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase. https://doi.org/10.1101/2021.01.05.425498

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