Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Lee, Kyung Suk; Balci, Hamza; Jia, Haifeng; Lohman, Timothy M.; Ha, Taekjip

doi:10.1038/ncomms2882

Cited by 97 publications

(118 citation statements)

References 60 publications

(107 reference statements)

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“…However, UvrD monomers show little helicase activity (12,16,21,24,25). In the absence of accessory proteins, helicase activity in vitro requires formation of a UvrD dimer (16,21,(25)(26)(27). The dimer unwinds dsDNA with rates of ∼70 base pair (bp) s −1 (20,21,24,27).…”

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confidence: 99%

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Large Domain Movements Upon UvrD Dimerization and Helicase Activation

Nguyen

Ordabayev

Sokoloski

et al. 2018

Biophysical Journal

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Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.single molecule | conformational heterogeneity | DNA motors | DNA repair U vrD from Escherichia coli is a nonhexameric SF1A DNA helicase/translocase, structurally similar to E. coli Rep and Bacillus stearothermophilus PcrA, which is involved in many aspects of genome maintenance (1, 2), including DNA repair (3, 4), replication (5-8), and recombination (7, 9-11). Its ATPdependent activities include translocation along single-stranded (ss) DNA (12-16), duplex DNA unwinding (17-22), displacement of RecA filaments from ssDNA (10, 11), and pushing of proteins along ssDNA (23).The activities of UvrD can be modulated in vitro by its assembly state and/or by binding partners (1). UvrD monomers can translocate directionally (3′ to 5′) along ssDNA [∼190 nucleotides (nts) s −1 ] (12, 13, 15, 16), in a reaction that is tightly coupled to ATP hydrolysis (14). However, UvrD monomers show little helicase activity (12,16,21,24,25). In the absence of accessory proteins, helicase activity in vitro requires formation of a UvrD dimer (16,21,(25)(26)(27). The dimer unwinds dsDNA with rates of ∼70 base pair (bp) s −1 (20,21,24,27). Rep and PcrA monomers also show no helicase activity and require oligomerization or an accessory protein for helicase activity in vitro (28-31).UvrD, Rep, and PcrA all contain four subdomains (1A, 2A, 1B, and 2B) (2,(32)(33)(34)(35), and the 2B subdomain can undergo a substantial rotation about a hinge region connecting it to the 2A subdomain (2,22,(32)(33)(34)(35)(36)(37). A structure of an apo UvrD monome...

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“…Its ATPdependent activities include translocation along single-stranded (ss) DNA (12)(13)(14)(15)(16), duplex DNA unwinding (17)(18)(19)(20)(21)(22), displacement of RecA filaments from ssDNA (10,11), and pushing of proteins along ssDNA (23).…”

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confidence: 99%

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

Nguyen

Ordabayev

Sokoloski

et al. 2018

Biophysical Journal

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“…Estimates of 193 nt/s and 1260 nucleotides for these parameters have been obtained from the analysis of the results of single-molecule measurements of UvrD translocation (46). Furthermore, the results of these single-molecule experiments suggest that the four to five nucleotides kinetic step-size for ssDNA translocation by UvrD cannot be attributed to molecular heterogeneity within the UvrD population (46); i.e., this kinetic stepsize cannot be explained by the presence of a distribution of translocation activities (rates, step-sizes, etc) for the UvrD enzymes.…”

Section: Sf1 Family Helicasesmentioning

confidence: 99%

All motors have to decide is what to do with the DNA that is given them

Briggs

Fischer

2014

Biomolecular Concepts

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DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions.

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“…To eliminate the distorting particle effects on biomolecular interaction this method was elaborated by substituting the particle with a fluorophore (tethered fluorophore motion-TFM) to observe the changes of the width of the fluorescence image (17). In our study, we exploit a different aspect of the TFM approach, namely, the dependence of the excitation intensity of a fluorophore on its position in the axial direction (18). In the TFM scheme, fluorophores of surface-immobilized biomolecules are excited by the evanescent electromagnetic field created by the total internal reflection (TIR) of the laser light off the glass-water interface (19).…”

Section: Introductionmentioning

confidence: 99%

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

Tutkus

Marčiulionis

Sasnauskas

et al. 2017

Biophysical Journal

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The single-molecule Fö rster resonance energy transfer (FRET) is a powerful tool to study interactions and conformational changes of biological molecules in the distance range from a few to 10 nm. In this study, we demonstrate a method to augment this range with longer distances. The method is based on the intensity changes of a tethered fluorophore, diffusing in the exponentially decaying evanescent excitation field. In combination with FRET it allowed us to reveal and characterize the dynamics of what had been inaccessible conformations of the DNA-protein complex. Our model system, restriction enzyme Ecl18kI, interacts with a FRET pair-labeled DNA fragment to form two different DNA loop conformations. The DNA-protein interaction geometry is such that the efficient FRET is expected for one of these conformations-''antiparallel'' loop. In the alternative ''parallel'' loop, the expected distance between the dyes is outside the range accessible by FRET. Therefore, ''antiparallel'' looping is observed in a single-molecule time trajectory as discrete transitions to a state of high FRET efficiency. At the same time, transitions to a high-intensity state of the directly excited acceptor fluorophore on a DNA tether are due to a change of its average position in the evanescent field of excitation and can be associated with a loop of either ''parallel'' or ''antiparallel'' configuration. Simultaneous analysis of FRET and acceptor intensity trajectories then allows us to discriminate different DNA loop conformations and access the average lifetimes of different states.

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Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Cited by 97 publications

References 60 publications

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

All motors have to decide is what to do with the DNA that is given them

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

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