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

• Published in: Nature communications Vol. 12; no. 1; pp. 7015 - 14
• Main Authors: Carney, Sean P., Ma, Wen, Whitley, Kevin D., Jia, Haifeng, Lohman, Timothy M., Luthey-Schulten, Zaida, Chemla, Yann R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2021; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/2204/2112; 631/45/612/1229; 631/535/1267; 631/57/2265; 631/57/2266; Biophysics; Catalysis; Conserved sequence; Deoxyribonucleic acid; DNA; DNA helicase; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA, Single-Stranded; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Estimates; Humanities and Social Sciences; Hydrolysis; Kinetics; Molecular dynamics; Molecular Dynamics Simulation; multidisciplinary; Nucleotide sequence; Optical Tweezers; Science; Science (multidisciplinary); Simulation; Single-stranded DNA; Strands; Translocation; Unwinding
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-27304-6

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. UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles.