Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer

Abstract

Single-molecule FRET is widely used to study helicases by detecting distance changes between a fluorescent donor and an acceptor anchored to overhangs of a forked DNA duplex. However, it has lacked single-base pair (1-bp) resolution required for revealing stepping dynamics in unwinding because FRET signals are usually blurred by thermal fluctuations of the overhangs. We designed a nanotensioner in which a short DNA is bent to exert a force on the overhangs, just as in optical/magnetic tweezers. The strategy improved the resolution of FRET to 0.5 bp, high enough to uncover the differences in DNA unwinding by yeast Pif1 and E. coli RecQ whose unwinding behaviors cannot be differentiated by currently practiced methods. We found that Pif1 exhibits 1-bp-stepping kinetics, while RecQ breaks 1 bp at a time but sequesters the nascent nucleotides and releases them randomly. The high-resolution data allowed us to propose a three-parameter model to quantitatively interpret the apparently different unwinding behaviors of the two helicases which belong to two superfamilies.Helicases are motor proteins involved in almost every aspect of nucleic acid metabolism [1][2][3][4]. When a helicase is loaded onto one of the overhangs (i.e., the tracking strand) of a forked DNA duplex and unwinds it, two nucleotides would be released per base pair unwound, leading to an increase in end-to-end distance of the overhangs. It is of great interest to know how a helicase uses the discrete energy derived from NTP hydrolysis to unwind DNA. The question remained unanswered for most helicases due to the lack of proper methods that can interrogate the stepping kinetics of helicases [5][6][7]. Optical tweezers (OT) are so far the most reliable technique to study single helicases with 0.5-bp resolution [8][9][10]. However, high-resolution measurements with OT require complicated instrumentation that is accessible to few laboratories only. In addition, OT measures one molecule at a time so that the throughput is usually very low. smFRET is a high-throughput technique for helicase assays. Using wide-field fluorescence microscopy, one can routinely record signals in parallel from hundreds of single molecules tethered to a surface [11][12][13][14]. To date,

Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase

“…Using a modified version of the n -step model (Figure 4C), global fitting of the unwinding kinetics of the gc36, gc48 and gc79 substrates using an integer series of n ranging from 1 to 7 revealed smallest χ v 2 values for an apparent kinetic step size of 5 bp for both RecQ and RecQ-dH (Figure 4B–D) with all DNA substrates, similar to that suggested by our MT results (Figure 3A–B) and by previous findings (Lin et al, 2017; Harami et al, 2015). However, the n -step model does not consider the sequence dependence of the rates of elementary unwinding steps, precluding the distinction between different microscopic mechanisms producing the same kinetic step size.…”

“…Based on previous studies of helicases (Manosas et al, 2010; Cheng et al, 2007; Neuman et al, 2005; Cheng et al, 2011; Lin et al, 2017; Myong et al, 2007), we considered two scenarios for RecQ-dH unwinding with an n -bp kinetic step size: either the enzyme unwinds n base-pairs simultaneously then rapidly translocates along the unwound DNA (simultaneous melting model), or it sequentially unwinds n base-pairs then releases the newly melted ssDNA (delayed release model) (Figure 3A). We exclusively simulated RecQ-dH unwinding and pausing kinetics rather than RecQ WT due to the significantly more complex behavior of the RecQ WT unwinding trajectories (Figure 1B).…”

“…However, we found that the pause durations were generally longer than would be expected for melting of 1 base pair when we compared our results with simulations. We considered two different scenarios: RecQ either destabilizes multiple base-pairs (≥2 bp) during each kinetic step similar to NS3 helicase (Cheng et al, 2007) or delays releasing of multiple unwound base-pairs similar to speculative models suggested in previous studies (Cheng et al, 2011; Lin et al, 2017; Myong et al, 2007; Ma et al, 2018). However, the minimal dependence of the unwinding rate on Na + concentration in addition to the sequence-dependent pauses cannot be explained by multi-base-pair melting.…”

A versatile platform for single-molecule enzymology of restriction endonuclease

“…[34][35][36][37] Speci¯cally, single-molecule F€ orster resonance energy transfer (sm-FRET) is widely applied for studying the structure, dynamics and function of proteins at the single-molecule level in real-time. [38][39][40][41] In typical sm-FRET studies, a pair of donor-acceptor°uorescent dyes is introduced to suitable positions of the protein or nucleic acid structure. The distance change of the two labeled positions can be monitored by the FRET e®ect of the two°uorophores with nanometer resolution in situ, making it a perfect tool for enzymology.…”

“…The distance change of the two labeled positions can be monitored by the FRET e®ect of the two°uorophores with nanometer resolution in situ, making it a perfect tool for enzymology. [41][42][43][44] However, these studies relied either on the speci¯c labeling of enzyme and the substrate using°uorescent dyes [38][39][40][41][42][43][44] or on the intrinsic°uorescence of the products. 45 The former method needs tedious°u orophore labeling and is technically di±cult to implement to di®erent systems.…”