Velocity and processivity of helicase unwinding of double-stranded nucleic acids

Betterton, Meredith D.; Jülicher, Frank

doi:10.1088/0953-8984/17/47/015

Cited by 17 publications

(34 citation statements)

References 65 publications

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“…The mean residence time of NS3 on RNA-AG is 2.70 Ϯ 0.02 s as compared with 2.00 Ϯ 0.02 s on RNA-10GC. The effect of strong barriers on the residence time of NS3 on RNA suggests an active interaction between NS3 and the barrier, which for stronger barriers leads to the accelerated detachment of the helicase (24). Indeed, the rate coefficient of NS3 dissociation upon unwinding G⅐C sequences is 0.5 Ϯ 0.2 s Ϫ1 at 7 pN, which is 2-fold faster than that on A⅐U sequences at the same force.…”

Section: Resultsmentioning

confidence: 99%

“…This conclusion would be valid for either a passive or an active helicase. In particular, for a passive helicase that contacts the ssRNA region and waits opportunistically for the fraying of the duplex without actively interacting with it, the mean residence time (i.e., the average time that the helicase stays on the substrate before detachment) should be the inverse of the helicase off-rate on ssRNA and independent of the position and magnitude of the barriers in the substrate (24). This is, however, not the case for NS3; rather, the residence time of NS3 on RNA depends on the presence of a barrier as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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RNA helicases regulate virtually all RNA-dependent cellular processes. Although much is known about helicase structures, very little is known about how they deal with barriers in RNA and the factors that affect their processivity. The hepatitis C virus encodes NS3, an RNA helicase that is essential for viral RNA replication. We have used optical tweezers to determine at the single-molecule level how the local stability of the RNA substrate affects the enzyme rate of strand separation, whether separation occurs by an active or a passive mechanism, and whether processivity is affected. We show that sequence barriers in RNA modulate NS3 activity. NS3 processivity depends on barriers ahead of the opening fork. Our results rule out a model where NS3 passively waits for the thermal fraying of double-stranded RNA. Instead, we find that NS3 destabilizes the duplex before separating the strands. Failure to do so before a strong barrier leads to helicase dissociation and limits the processivity of the enzyme.hairpin ͉ molecular motors ͉ optical tweezers ͉ processivity A wide range of RNA metabolic activities (1) require the breaking of base pairs in double-stranded RNA (dsRNA) by RNA helicases. Despite recent progress in the study of these motor proteins (2-4), the physical mechanisms by which they move and catalyze the strand separation are not well understood. Helicase models ranging from a pure Brownian ratchet to a pure power stroke action have been discussed (5-8), but experimental data to support them for RNA helicases have been lacking. The hepatitis C virus NS3 protein is a superfamily 2, 3Ј to 5Ј RNA helicase (9) known to be essential for virus replication and, thus, an antiviral drug target (10). Recently, NS3 has been the subject of single-molecule manipulation studies (4), which revealed that NS3 makes steps of 11 bp each made up of three substeps (4). RNA molecules contain various kinetic barriers to mechanical unfolding (11). How these barriers influence the activity of motor proteins that work on RNA will depend on the mechanism of the motor. To probe the molecular mechanism of NS3 unwinding activity, we have designed and characterized RNA molecules with various mechanical unfolding barriers and used single-molecule methods to investigate how these barriers affect the velocity, pausing, and processivity of the helicase in real time. ResultsDesign and Characterization of RNA Hairpin Substrates. We first designed two RNA hairpin substrates that terminate both on a tetraloop (Fig. 1A) to monitor the response of a single NS3 helicase to weak and strong sequence barriers. Substrate RNA-AG has 30 A⅐U pairs followed by 30 G⅐C pairs; RNA-GA has the A⅐U and G⅐C sequences interchanged. The sequences within A⅐U and G⅐C regions were chosen to minimize the formation of alternative secondary structures other than the desired 60-bp hairpin. In our experiment, a single RNA hairpin was attached between a microsphere in an optical trap and a microsphere placed on the end of a micropipette through hybrid RNA-DNA handles to se...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Cheng¹,

Dumont

Tinoco³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

149

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“…Recently, a phase coexistence based mechanism for helicase activity has been proposed by Bhattacharjee and Seno [7]. A kinetic model has also been proposed recently [8] while a random walk model was used in an earlier study [9] to analyze the movement on DNA. The phase-coexistence mechanism is based on the unzipping phase transition of a ds-DNA by a force, which was first shown in a continuum model in Ref.…”

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confidence: 99%

Helicase activity on DNA as a propagating front

Bhattacharjee¹

2004

Europhys. Lett.

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We develop a propagating front analysis, in terms of a local probability of zipping, for the helicase activity of opening up a double stranded DNA (dsDNA). In a fixed-distance ensemble (conjugate to the fixed-force ensemble) the front separates the zipped and unzipped phases of a dsDNA and a drive acts locally around the front. Bounds from variational analysis and numerical estimates for the speed of a helicase are obtained. Different types of helicase behaviours can be distinguished by the nature of the drive.

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“…By generalizing the original mathematical framework introduced by Betterton and Jülicher [29][30][31], we predicted that the processivity of active and passive helicases should always increase in response to external forces applied to the termini of dsDNA or dsRNA. Unlike the processivity, the velocity of unwinding, however, exhibits no such universal behavior, and exhibit a variety of responses to external force depending on whether the helicase is active or passive [28].…”

Section: Introductionmentioning

confidence: 99%

“…Our theory also shows that an externally applied force, which in vivo could be generated by binding of single stranded partner proteins, results in an increase in processivity in all helicases, regardless of whether they are active or passive. [29,31] that accounts for the finite processivity of the helicase, allows for an arbitrary step-size, and a general interaction range between the helicase and the double strand. Fig 1a shows the helicase (red filled circles) as it translocates on the ss nucleic acid strand (depicted as a bold black line).…”

Section: Introductionmentioning

confidence: 99%

Processivity, velocity and universal characteristics of nucleic acid unwinding by helicases

Chakrabarti

Jarzynski

Thirumalai

2018

Preprint

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Helicases that act as motors and unwind double stranded nucleic acids are broadly classified as either active or passive, depending on whether or not they directly destabilize the double strand. By using this description in a mathematical framework, we derive analytic expressions for the velocity and run-length of a general model of finitely processive helicases. We show that, in contrast to the helicase unwinding velocity, the processivity exhibits a universal increase in response to external force. We use our results to analyze velocity and processivity data from single molecule experiments on the superfamily-4 ring helicase T7, and establish quantitatively that T7 is a weakly active helicase. We predict that compared to single-strand translocation, there is almost a two ordersof-magnitude increase in the back-stepping probability of T7 while unwinding double-stranded DNA. Our quantitative analysis of T7 suggests that the tendency of helicases to take frequent back-steps may be more common than previously anticipated, as was recently shown for the XPD helicase. Finally, our results suggest the intriguing possibility of a single underlying physical principle governing the experimentally observed increase in unwinding efficiencies of helicases in the presence of force, oligomerization or partner proteins like single strand binding proteins. The clear implication is that helicases may have evolved to maximize processivity rather than speed.

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Velocity and processivity of helicase unwinding of double-stranded nucleic acids

Cited by 17 publications

References 65 publications

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork

Helicase activity on DNA as a propagating front

Processivity, velocity and universal characteristics of nucleic acid unwinding by helicases

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