Abstract
UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. 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.
Highlights
UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex
Helicases are a class of molecular motors that use nucleoside triphosphate (NTP) hydrolysis to translocate on single-stranded nucleic acids (NA) and unwind double-stranded NA1,2
We calculated the number of base pairs unwound over time from the change in extension of the tether as UvrD released the newly formed ssDNA, converting each 2 nucleotides released into 1 bp unwound
Summary
UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. 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. Crystal structures of UvrD and its homolog PcrA on a ss/dsDNA junction have been used to infer an unwinding step size of 1 base pair (bp) unwound per ATP hydrolyzed for UvrD and a translocation step size of 1 nucleotide (nt) per ATP for PcrA17,19 This value agrees with ensemble kinetic measurements of the chemical step size (or ATP coupling ratio)—the distance advanced for each ATP hydrolyzed2,3—of 1 nt/ATP during ssDNA translocation by both helicases[20,21]
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