Branched unwinding mechanism of the Pif1 family ofDNA helicasesSaurabh P. Singha, Andrea Sorannoa,b, Melanie A. Sparksa, and Roberto Gallettoa,1

aDepartment of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110; and bCenter for Science andEngineering of Living Systems, Washington University in St. Louis, St. Louis, MO 63130

Edited by Kevin D. Raney, University of Arkansas for Medical Sciences, Little Rock, AR, and accepted by Editorial Board Member Stephen J. Benkovic October24, 2019 (received for review September 10, 2019)

Members of the Pif1 family of helicases function in multiple pathwaysthat involve DNA synthesis: DNA replication across G-quadruplexes;break-induced replication; and processing of long flaps duringOkazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNAreplication across arrays of proteins tightly bound to DNA. This is asurprising feat since DNA rewinding or annealing activities limit theamount of single-stranded DNA product that Pif1 can generate,leading to an apparently poorly processive helicase. In this work,using single-molecule Förster resonance energy transfer approaches,we show that 2 members of the Pif1 family of helicases, Pif1 fromSaccharomyces cerevisiae and Pfh1 from Schizosaccharomycespombe, unwind double-stranded DNA by a branched mechanismwith 2 modes of activity. In the dominant mode, only shortstretches of DNA can be processively and repetitively opened, withreclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches ofDNA are unwound via a path that is separate from the one leadingto repetitive unwinding. Analysis of the kinetic partitioning be-tween the 2 different modes suggests that the branching pointin the mechanism is established by conformational selection, con-trolled by the interaction of the helicase with the 3′ nontranslocatingstrand. The data suggest that the dominant and repetitive modeof DNA opening of the helicase can be used to allow efficient DNAreplication, with DNA synthesis on the nontranslocating strandrectifying the DNA unwinding activity.

single molecule | DNA unwinding | helicase | DNA replication

Growing experimental evidence indicates that multiple heli-cases/translocases display a peculiar behavior in which a

single motor protein molecule can repetitively translocate onnucleic acid (1–6) or unwind double-stranded DNA (7–11) andG-quadruplex structures (6, 12–14). How this repetitive enzy-matic activity is used for function remains unclear, but it may belinked to the specific pathway in which these helicases operate,the nature of the substrate itself, and the directionality of un-winding. Repetitive unwinding activity may be of particular sig-nificance for 5′-3′ helicases, such as those of the SF1B Pif1 family(15, 16), whose activity may be coupled to DNA replication. Pif1from Saccharomyces cerevisiae, the best studied of this class ofhelicases and its founding member, displaces telomerase fromtelomeric ends (17–22), facilitates DNA replication at G-quadruplex–forming sequences during lagging strand DNA syn-thesis (23–26), cooperates with Dna2 helicase/nuclease in longflap processing during Okazaki fragment maturation (27–31),and functions with DNA polymerase δ during break-inducedDNA replication (32–35). Similarly, Pfh1, the Pif1 homologin Schizosaccharomyces pombe, functions in many of the samepathways and has biochemical activities similar to Pif1 (36–42).Single-molecule experiments showed that Pif1 can repetitively

reel in single-stranded DNA (ssDNA) or unwind G-quadruplexsubstrates (6, 12, 43). Repetitive unwinding of G-quadruplexeshas been proposed to be a means to keep the formation of thesestructures under check (6). Also, once stable G-quadruplexes are

formed, these can pose a significant obstacle to DNA replication,and the unwinding activity of Pif1 or Pfh1 is needed to allowsignificant DNA synthesis (44). Intramolecular G-quadruplexesare compact secondary structures held by a well-defined numberof Hoogsteen base pairs, and limited unwinding by the helicasemay be sufficient to lead to their full opening. However, thesituation can be different for double-stranded DNA (dsDNA),where, depending on the length of the duplex region, repetitivehelicase activity may not lead to full unwinding of the substrate.Indeed, the intrinsic dsDNA unwinding activity of Pif1 is counter-acted by DNA rewinding or annealing activities (45, 46) that limitthe amount of ssDNA product generated and lead to an appar-ently poor helicase. Moreover, under applied force, Pif1 displays acomplex unwinding mechanism in which dsDNA unwinding iscounteracted by either the helicase sliding back on the substrate orstrand-switching and translocating back (47). In the absence ofapplied force, it remains unclear how these different activitiescontribute to the dsDNA unwinding mechanism of Pif1. In light ofall these activities that compete with dsDNA unwinding, it issurprising that, when coupled to DNA replication, the helicaseactivity of Pif1 is enough to lead to kilobases of DNA synthesis anddisplacement of proteins tightly bound to dsDNA (32, 33, 48). It ispossible that DNA synthesis on the nontranslocating strand maybe a means to suppress DNA rewinding by Pif1, favoring efficientunwinding and removal of protein blocks (45, 48). In this scenario,one may expect that, when coupled to DNA synthesis, Pif1 doesnot need to operate as a processive helicase that can open largeamounts of DNA. Rather, efficient and processive opening ofshort stretches of DNA is all that this class of helicases may needto achieve to allow synthesis on the 3′ nontranslocating strand toproceed efficiently. If this were the case, one would expect the

Significance

DNA helicases are motor proteins that operate on nucleic acidsduring DNA replication, recombination, and repair. Repetitivetranslocation on and unwinding of nucleic acids is an emergingproperty of multiple helicases. How this repetitive enzymaticactivity may be used for function remains unclear. For the 5′-3′Pif1 family of helicases our data suggest a model in which ahighly processive opening of short stretches of DNA is all thatthis class of nonreplicative helicases need to achieve whentheir function is coupled to DNA replication.

Author contributions: S.P.S., A.S., and R.G. designed research; S.P.S., A.S., M.A.S., and R.G.performed research; S.P.S., A.S., M.A.S., and R.G. analyzed data; and S.P.S., A.S., and R.G.wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission. K.D.R. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1To whom correspondence may be addressed. Email: galletto@wustl.edu.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1915654116/-/DCSupplemental.

First published November 19, 2019.

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DNA unwinding mechanism of these helicases to be dominated bythis latter mode of unwinding, rather than by processive unwindingin sensu stricto. In this work, we set out to test this possibility andused single-molecule Förster resonance energy transfer (smFRET)approaches to study in real time the unwinding mechanism ofS. cerevisiae Pif1 and S. pombe Pfh1.

ResultsIndividual Molecules of Pfh1 Exhibit Repetitive Attempts of PartialdsDNA Unwinding, Composed of Multiple ATP-Dependent Steps. Inensemble experiments, ATPase active full-length Pfh1 helicasefrom S. pombe has limited intrinsic dsDNA unwinding activity inthe absence of a trap to prevent reannealing of the unwoundstrands (SI Appendix, Fig. S1). This behavior is similar to the onereported for Pif1 (45, 46) and is consistent with a rewindingactivity counteracting unwinding (45). In order to study the in-terplay between these 2 activities in real time, based on ourprevious ensemble work (45) we designed smFRET experimentsusing DNA substrates labeled with donor-acceptor couples atdifferent positions (SI Appendix, Fig. S2). The substrates containa short biotin-labeled 18-bp dsDNA handle for attachment to a

pegylated surface via a biotin-neutravidin-biotin sandwich, a30-bp dsDNA region to be unwound, a 5′ oligo-dT10 tail as an entrypoint for the helicase, and variations in the 3′-tail (Fig. 1A). Inthe experiments, Pfh1 is loaded onto surface immobilized DNAs,and excess enzyme is removed by extensive washing. Upon ad-dition of ATP, but not its nonhydrolyzable analogs ATPγS orAMP-PNP, the Cy3 and Cy5 intensities show clear anticorrelatedchanges and the corresponding FRET signal transitions betweenhigh and low values, consistent with Pfh1 opening the dsDNAwithout full unwinding (Fig. 1B). Indeed, at 10 μM ATP with thisDNA substrate that contains a 3′-dT22 tail only 7% of the DNAmolecules show evidence of unwinding (SI Appendix, Fig. S3A),while the majority undergo a series of repetitive transitions be-fore Pfh1 dissociates or the fluorophores photobleach. This be-havior does not arise from additional Pfh1 molecules bound tothe 22-nucleotide (nt)-long 3′-tail, as shown in experimentswhere the 3′-tail is substituted with either a 21-bp duplex regionor a 3′-dT2 tail (SI Appendix, Fig. S3 C and D). Furthermore,immobilization of Pfh1 on the surface reveals that the observedATP-dependent FRET transitions originate from a Pfh1 mono-mer (SI Appendix, Fig. S3E). Importantly, ATP-dependent FRET

Fig. 1. Individual Pfh1 molecules display repetitive attempts of partial dsDNA unwinding. (A) Schematic of the smFRET experiment with D-A at thejunction. (B) Representative anticorrelated changes in Cy3 and Cy5 intensities and corresponding FRET changes after starting the reaction with addition ofATP. For clarity, a 15-point moving average is overlaid with the raw data. (C and D) Distribution of FRET states visited during repetitive unwinding of thesubstrate with D-A at the junction in A or 20 bp apart in D. Solid lines are the fits to a Gaussian distribution. The white line indicates the distribution ofFRET values before addition of ATP. (E ) ATP dependence of the difference in mean FRET values of the closed and open states. (F–I) Distribution of thetimes spent in the different states during the repetitive cycle for the substrate with D-A at the junction and 10 μM ATP. The lines are fits to either a singleexponential function or a gamma function with 2 equal rates. (J) Minimal kinetic model that combines the information from each step during the re-petitive cycle of partial unwinding.

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transitions, from low to high, are observed also with a DNAsubstrate in which Cy5 is positioned 20 bp into the duplex region(SI Appendix, Fig. S3B), confirming that the dsDNA is beingpartially unwound. Analyses of experiments with Cy3 and Cy5placed at different positions within the substrate or only Cy3 po-sitioned 20 bp into the duplex strongly suggest that the processiveand repetitive unwinding is limited to short stretches of dsDNA(<20 bp) (SI Appendix, Fig. S4) and that this is a property that origi-nates from the helicase core of the enzyme (SI Appendix, Fig. S5).Repetitive unwinding/translocation activity on DNA, monitored

by FRET and/or protein-induced fluorescence enhancementchanges, has been reported for multiple helicases/translocases (1,3, 4, 7, 8, 10, 13, 14, 49), including Pif1 (6, 12, 43, 47). The re-petitive cycling behavior has been shown to be ATP dependent, asexpected for a phenomenon driven by a motor protein; however,how ATP affects the multiple steps within the cycle has not beenextensively characterized. To this end, we note that, during therepetitive cycles of partial DNA unwinding, the system appears tospend most of its time in 4 states: 2 well-defined states associatedwith high and low transfer efficiency and 2 transitions (from highto low and from low to high FRET) that connect these states. Weidentify these states by clustering the data based on their Euclid-ean distance, and, for each molecule, we estimate the transferefficiency of the closed and open states, the dwell time in these 2states, and the duration of the unwinding or rewinding transitions(SI Appendix, Fig. S6).For the DNA construct with the donor-acceptor (D-A) pair at

the junction, analysis of 225 molecules and ∼4,900 transitionsshows that at 10 μM ATP the system transitions from a closedstate with mean transfer efficiency (E = 0.77 ± 0.09) to an openstate with a lower mean transfer efficiency (E = 0.4 ± 0.1) (Fig.1C). Importantly, the system transitions from the open state backto a closed state that is the same as the Pfh1-DNA complexbefore addition of ATP (E = 0.78 ± 0.08), indicating that theentire dsDNA region that was opened has been reclosed. Thesame is true when the donor and acceptor are 20 bp apart (Fig.1D). Moreover, independent of whether FRET is monitoredwith the acceptor at the junction or at 20 bp into the dsDNA, thedifference in the average FRET value between the closed andopen states does not change as a function of ATP concentration(Fig. 1E), indicating that on average the system opens the samenumber of base pairs before returning to its original closed state.The times spent in the closed and open states are exponen-

tially distributed (Fig. 1 F and G, and SI Appendix, Fig. S7 A andB), indicating that the system escapes from a single state, with noevidence of hidden intermediates. In contrast to the times spentin the closed and open states, the times of unwinding are notexponentially distributed. Rather, a gamma function with 2 stepsof equal rate is sufficient to describe the distribution of the un-winding times (Fig. 1H and SI Appendix, Fig. S7C), independentof the position of the donor-acceptor couple. Such a distributionof times is indicative of the presence of an intermediate unwoundstate that precedes formation of the open complex. Similarly, therewinding times do not appear to follow an exponential distribu-tion (Fig. 1I and SI Appendix, Fig. S7D), suggesting the presenceof a hidden intermediate. However, these events approach thetime resolution of recording (30 Hz), and their number may beunderestimated. Thus, assuming that the events in the first 2 timebins are within the time resolution limit of the measurement, thedistribution of the rewinding times was fitted to an exponentialfunction. The calculated rewinding rates show little ATP depen-dence (SI Appendix, Fig. S7H), suggesting that either the rewindingstep is ATP independent or, if there is an ATP dependence, itmust occur at lower ATP concentrations. Combining the infor-mation from analysis of the ATP dependence of each individualphase during the cycle of opening attempts (SI Appendix, Fig.S7 E–G) leads to the minimal kinetic model depicted in Fig. 1J.

Finally, we performed the same measurements for the Pif1helicase (SI Appendix, Figs. S8 and S9). Pif1 behaves similarly toPfh1 with the difference that the lifetimes of the different statesappear to be slightly shorter. Shorter lifetimes of the closed andopen state suggest either a higher affinity of Pif1 for ATP or afaster rate of escape to the next step. Also, the unwinding times forPif1 are slightly faster than for Pfh1, suggesting that Pif1 is a fasterhelicase. However, at difference with Pfh1, the rewinding times forPif1 appear to have some ATP dependence, as would be expectedif this step were driven by the motor protein activity of Pif1.

During Rewinding Neither Pfh1 nor Pif1 Strand-Switch. Strand-switching during unwinding has been proposed for differentDNA helicases as a mechanism that leads to rezipping of theopened dsDNA (7, 11, 50). Recent single-molecule work, underapplied force, provides evidence of a complex mechanism ofunwinding by Pif1 and suggests that during unwinding Pif1 canswitch to the opposite DNA strand, leading to DNA rewinding(47). Because this mechanism depends on the translocation ac-tivity of Pif1 on ssDNA, an ATP dependence is expected. Basedon our observation that, for Pfh1, the rewinding step from theopen to the closed state does not show an ATP dependence, weconclude that, for Pfh1, strand-switching does not significantlycontribute to this step. The same may not be the case for Pif1,since it shows some ATP dependence for rewinding.In order to provide a more direct test of whether strand-

switching may contribute to rewinding of the DNA during thecycle of partial unwinding, we designed a simple experimentalstrategy. Our idea is based on the fact that Pif1 can unwindRNA-DNA hybrids only when the strand used for translocationis DNA and not RNA (51), indicating that the RNA strandcannot be used for translocation. Because the basic tenet of astrand-switching mechanism is translocation of the helicase onthe opposite strand, one would expect that, if this strand wereRNA, no FRET transitions should be observed. Therefore, weperformed experiments with both Pfh1 and Pif1 and RNA-DNAhybrids with Cy3-Cy5 at different positions along the substrate.Independent of the position of the donor-acceptor couple,FRET transitions can be clearly observed for both Pfh1 and Pif1(Fig. 2 A–D), as well as for a Pfh1 variant missing its first 291amino acids (SI Appendix, Fig. S5C). These data provide directexperimental evidence that under our assay conditions strand-switching is not the major reason why, during the cycle of partialunwinding, the DNA rezips.Interestingly, for Pfh1 and the RNA-DNA hybrid with the

donor-acceptor at the junction, the ATP dependence of thelifetime in the different states of the repetitive cycle of FRETtransition is similar to the one observed for dsDNA and a 3′-dT22tail (Fig. 2E). This result strongly suggests that the 3′-tail of thesubstrate does not have a significant role in regulating the ki-netics of repetitive transition from the closed to the open states.This point is further reinforced by the observation that neitherthe length (from 5 to 22 nt) nor the nature (oligo-dT, mixedsequence, dsDNA) of the 3′-tail significantly modulates the ki-netics of the repetitive FRET transitions (Fig. 2E).

The Repetitive DNA Opening Attempts Are on a Separate Pathwayfrom Full Unwinding.Although the 3′-tail of the substrate does notappear to modulate the kinetics of the repetitive FRET transi-tions, we found that, in the presence of a 3′-tail of mixed se-quence composition (3′-ss22), the fraction of molecules that isfully unwound by Pfh1 increases. For example, at 10 μM ATP of420 DNA molecules that show Pfh1-dependent activity, 58%show evidence of full unwinding of the dsDNA (Fig. 3 A and B).A significant fraction of full unwinding occurs also with a 3′-ss5tail, in stark contrast to the limited unwinding observed with a 3′-dT22 tail (Fig. 3B). Also, an effect of the nature of the 3′-tail ofthe substrate is evident in experiments monitoring unwinding via

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disappearance of Cy3 signal from DNA substrates immobilizedon a surface (SI Appendix, Fig. S10 A and B). This is the samephenomenon that we reported for Pif1 from ensemble studies(45), corroborated here by single-molecule experiments (SI Ap-pendix, Fig. S10C), and indicates that this is a general property ofmembers of the Pif1 family of helicases.These observations raise the question of how the nature of the

3′-tail of the DNA modulates the probability of unwinding and,importantly, whether the multiple partial unwinding attempts areon-pathway with formation of full-length unwound product orabortive off-pathway events. To this end, we note that the un-winding behavior of Pfh1 can be divided in 2 classes. For ex-ample, at 10 μM ATP, 50% of the molecules show repetitiveFRET transitions followed by the presence of a clear final openintermediate state before full unwinding occurs (class 1), while8% show the presence of this intermediate without evidence ofFRET transitions preceding it (class 2) (Fig. 3 A and C). Im-portantly, the fraction of molecules that unwinds the substratewith class 2 behavior increases as the ATP concentration in-creases (Fig. 3D). Furthermore, the presence of 2 classes ofunwinding behavior for Pfh1 does not depend on the position ofthe donor-acceptor couple (SI Appendix, Fig. S11 A and B) or thelength of the 3′-tail (Fig. 3D) and is a property shared with Pif1(SI Appendix, Fig. S11C).

The presence of 2 distinct classes of unwinding behaviorprovides evidence that the repetitive cycle of partial unwinding isnot on-pathway with complete unwinding (model 1 in Fig. 4A).To test this point further, we calculated the average number ofcycles that the system undergoes before unwinding (Fig. 3E andSI Appendix, Fig. S12). The average number of repetitive cyclesincreases slightly with increasing ATP concentration, indepen-dently of the length of the 3′-tail (Fig. 3 F and G). If the re-petitive unwinding attempts were on-pathway, the increase in thefraction of full unwinding as ATP increases would be expected tolead to a decrease in the number of cycles of partial unwindingattempts. Importantly, the same behavior is observed also whenthe donor is placed 20 bp into the dsDNA (SI Appendix, Fig.S13A) and with Pif1 as well (SI Appendix, Fig. S13B), arguingthat the phenomenon is conserved for both helicases.Next, we sought to identify the branching point at which the

system either enters an off-pathway cycle of repetitive unwindingattempts or commits to enter the open intermediate from whichfull unwinding occurs. Model 2 in Fig. 4A depicts a simplemechanism where the branching point is at the ATP-bound closedstate. Because in this model branching occurs from a single closedstate, we would expect the times spent in this state to be expo-nentially distributed. However, the times spent in the final closedstate are not exponentially distributed and can be fitted with agamma function with 2 steps of equal rate (Fig. 4B). Such a

EDifferent 3’-end:

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Fig. 2. Neither Pfh1 nor Pif1 strand-switch. (A–D) Representative examples of repetitive FRET transitions observed at 10 μM ATP for both Pfh1 and Pif1 withRNA-DNA hybrids and the D-A couple at the indicated positions. (E) ATP dependence of lifetime in the different stages of the repetitive FRET transitions forPfh1 and the DNA-RNA hybrid compared to dsDNAs with different 3′-tails.

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distribution of times is inconsistent with model 2 and stronglyargues for the presence of a second closed hidden intermediate.Models 3 and 4 in Fig. 4A depict 2 possible mechanisms wherebythe branching point originates from a second closed state. Themajor distinction between these 2 models is that in model 3 theequilibrium is between 2 ATP-bound closed states (i.e., a se-quential mechanism), while in model 4 the 2 closed states existin equilibrium before ATP binding (i.e., conformational selec-tion). In the sequential model 3 the equilibrium between 2ATP-bound closed states is a first-order transition and is notexpected to be ATP dependent. However, the rates calculated

from fitting the distribution of times spent in the final closedstate are clearly ATP dependent (Fig. 4C), lending strong supportto model 4.Lastly, the final open state is kinetically distinct from the open

state visited during the cycle of partial unwinding attempts. First,the average time spent in this state is longer than the time spentin the open state during the repetitive cycle (SI Appendix, Fig.S12A). Second, independent of the ATP concentration, thetimes spent in the final open state are not exponentially dis-tributed (Fig. 4D). These distributions can be fitted with agamma function with 7 to 10 steps, indicating that, upon escape

Fig. 3. Partitioning between partial dsDNA opening and full unwinding. (A) Representative repetitive FRET transition observed at 10 μM for Pfh1 and asubstrate with D-A at the junction and a 3′-tail composed of 22 nt of mixed sequence compositions. Full unwinding events can occur either after repetitiveattempts (class 1) or directly (class 2). (B) ATP dependence of the fraction of full unwinding by Pfh1 of substrates with D-A at the junction and different 3′-tails.(C) Examples of class 1 and class 2 unwinding behavior showing the changes in Cy3 and Cy5 intensities. (D) ATP dependence of the fraction of class 1 and class2 unwinding behaviors for a 3′-tail with 22 or 5 nt. (E) Schematic of 2 ways of calculating the average number of partial unwinding attempts before fullunwinding. (F and G) ATP dependence of the average number of partial unwinding attempts, accounting or not for the presence of tend.

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from the final open state, the dsDNA is unwound in multipleATP-dependent steps.

Partial DNA Opening Is aMode of Unwinding That Can Be Used during DNASynthesis. When coupled to DNA synthesis on the nontranslocatingstrand, both Pif1 and Pfh1 stimulate DNA replication through stableG-quadruplex structures (44). Moreover, the 5′-3′ unwindingactivity of Pif1 stimulates kilobase pair of strand-displacementDNA synthesis, even when proteins are tightly bound to dsDNA(48). Similarly, DNA primer extension assays using an exonuclease-deficient DNA polymerase δ (SI Appendix, Fig. S14), underconditions in which it has limited activity (52), also show thatPfh1 stimulates DNA synthesis (Fig. 5A). We note that Pfh1 isless efficient than Pif1 in stimulating DNA synthesis, as evi-denced by the higher enzyme concentration needed and theslower processing of intermediates and accumulation of fullproduct when an 80-bp dsDNA is used. This is consistent withPfh1 being a slower helicase and suggests that unwinding of theDNA rather than synthesis is the overall rate-limiting step inthe reaction.

The observation that DNA unwinding by Pfh1 and Pif1 occursvia 2 modes (one dominant, short ranged, and highly repetitive,the other less frequent and longer ranged) raises the questions ofwhether only one or both modes are functionally relevant duringDNA synthesis. We note that the probability of full unwinding,by either class 1 and 2, is modulated by the nature of the 3′nontranslocating strand (Fig. 3B). The presence of dsDNA at the3′ arm of the substrate does not affect the kinetics of repetitiveunwinding, as monitored via FRET transitions with the D-A coupleat the junction (21-bp dsDNA, Fig. 2E); however, we detected alimited amount of full unwinding. This suggests that, similar to3′-T22, with a 21-bp dsDNA at the 3′ arm the long-ranged modeof unwinding is not a frequent event, and the helicase activity isdominated by the short-ranged and repetitive mode. Consistentwith this, unwinding assays monitoring the disappearance of Cy3signal from DNA immobilized on the surface show limited un-winding by either Pfh1 or Pif1 (Fig. 5B). If only the long-rangedmode of unwinding were to be used, because of its low frequencyone may expect a limited effect of DNA synthesis on DNA un-winding. However, addition of DNA polymerase δ significantlyincreases the apparent rate and extent of full product formation

Model 1on-pathway

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(U) → O2→ → → unwound

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Fig. 4. The branch point in the mechanism is determined by conformational selection of an unwinding competent state. (A) Schematic of 4 possible kineticsschemes to explain the partitioning between abortive attempts and full unwinding. Model 1 is an on-pathway mechanism, while models 2 to 4 are off-pathway mechanisms with different branching points. (B) Distribution of the time spent by Pfh1 in the last closed state with a substrate with D-A at thejunction and 10 μM ATP. The solid line is the fit to a gamma function with 2 equal rates. (C) ATP dependence of the rate of escape from the last closed state.(D) Distribution of the time spent in the final intermediate state before full unwinding at the indicated ATP concentrations. The solid lines are fits to a gammafunction with 7 to 10 equal rates.

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(Fig. 5B), suggesting that the short-ranged mode of unwinding(e.g., partial opening) is being used.

DiscussionIn summary, we showed that the unwinding of dsDNA by the S.pombe Pfh1 helicase, like S. cerevisiae Pif1, is dominated byhighly processive and repetitive attempts of partial DNA open-ing. The presence of these abortive unwinding events explainsthe apparent DNA rewinding activity observed in ensemble

experiments: repetitive opening of a limited number of base pairs(e.g., <20 bp) would not lead to unwinding of sufficiently longdsDNA. Interestingly, Pif1 has been proposed to unwind dsDNAin 1-bp steps (53, 54), and our data clearly point to an inter-mediate state visited during unwinding. However, during thepartial unwinding attempts, both Pif1 and Pfh1 open more than2 bp, yet only one intermediate is kinetically populated. Therefore,this intermediate must originate from the opening of multiplebase pairs. Importantly, repetitive unwinding of dsDNA has been

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Fig. 5. Cooperation between DNA synthesis and helicase activity. (A) DNA primer extension assays were performed with exonuclease-deficient DNA poly-merase δ (Pol δDV), alone (black) or in the presence of Pfh1 (blue) or Pif1 (red). The reaction and substrates are schematically depicted, with the analyzedreplication products binned in different groups and their quantification reported in the corresponding graphs. Error bars are the SD from 3 independentexperiments. (B) Unwinding of Cy3-labeled DNA substrates immobilized on the surface by either Pfh1 or Pif1 in the absence or the presence of Pol δDV.Experiments to control for photobleaching or for the activity of Pol δDV alone are indicated.

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reported for other helicases, and multiple mechanisms thatwould lead to closure of the transiently opened dsDNA havebeen proposed. For example, strand-switching during un-winding, with the helicase being able to jump to the oppositessDNA strand and translocate back, has been proposed formultiple helicases (7, 11, 50, 55), including Pif1 (47). Theobservation in this work that, for both Pfh1 and Pif1, repetitiveunwinding occurs also on RNA-DNA hybrids provides strongexperimental evidence that strand-switching is not a significantmechanism leading to closure of the partially opened dsDNA.On the one hand, a spring-loaded or snap-back mechanism (1,8, 55), where the repetitive cycle of unwinding originates fromthe helicase remaining bound to a portion of the substrate,may explain closure of the partially opened DNA. While Pif1has been shown to repetitively reel in ssDNA or unwind G-quadruplexes when bound with high affinity to a 5′-ds/ssDNAjunction (6), neither ssDNA translocation nor dsDNA un-winding require such a site to occur (45, 56). For the DNAsubstrates in this work, the repetitive partial unwinding at-tempts occur independently of the 3′-ssDNA tail of the sub-strate, leaving the 5′-ssDNA as the potential anchor point. Inthis scenario, Pfh1 or Pif1 would have to remain bound to the10-nt 5′-tail as they unwind the downstream duplex. On theother hand, closure of the partially unwound DNA could bedue to the helicases slipping back on the substrate. This wouldbe consistent with the same mechanism reported for Pif1 as analternative pathway to strand-switching (47) and for otherhelicases (57–59). Although our data do not allow us to un-ambiguously discriminate between snap-back and slippageback, based on our observation that DNA synthesis on thenontranslocating strand stimulates DNA unwinding, we favorthe latter explanation. This is not to say that neither Pif1 norPfh1 can use either strand-switching or spring-loaded mecha-nisms. It is possible that these mechanisms may become sig-nificant under different experimental conditions, such as whenDNA is under tension (47) or a high-affinity site for the heli-cases is present (6).Importantly, the observation that Pfh1, Pif1, and other heli-

cases can undergo repetitive cycles of partial dsDNA unwindingraises the question of whether these attempts are on-pathway tofull enzymatic activity (e.g., full product formation) or off-pathway events. We showed here that for both Pfh1 and Pif1the latter appears to be the case. The data suggest a branchedmechanism of DNA unwinding, where the major branchingpoint is established by conformational selection of an un-winding competent complex. Such a mechanism would explainwhy the nature of the 3′-ssDNA tail of the substrate does notaffect the kinetics of partial DNA opening, but instead modu-lates the probability of full product formation. We propose thatinteraction of the 3′-tail of the substrate with a secondary siteon Pfh1 and Pif1 (45), separate from the primary interactionsite with the 5′ translocating strand, leads to a conformation ofthe enzymes that is capable of unwinding longer stretches ofdsDNA. The location of the secondary DNA site and how itmay interact with 3′-tails of different sequence compositionremain to be determined. Moreover, whether regulation ofunwinding by the nature of the 3′-ssDNA tail of the substrate isunique to Pif1-family members or shared with other helicases iscurrently unknown. Crystal structures of Pif1-family memberssuggest that the 2B domain undergoes rotation upon DNAbinding (60–63). We speculate that interaction of the 3′ non-translocating strand with a secondary DNA site may modulaterearrangements of the 2B domain that lead to different modesof unwinding. For example, stabilization in a closed state of the2B domain of Rep and PcrA helicases leads to highly processiveDNA unwinding (64).Finally, the observation that Pfh1 and Pif1 can switch between

2 modes of unwinding, one in which the enzyme processively

opens short stretches of DNA and the other in which longerDNA stretches can be unwound, raises the question of whetherboth modes are functional or only one is predominantly used.The ability of the nature of the 3′-ssDNA tail of the DNA toshunt the system toward unwinding of longer stretches of DNAcould be used to regulate, in a DNA-sequence–dependent con-text, the activity of these helicases. However, even in thismode the length of DNA that can be unwound remains limited.In cells, Pif1 and Pfh1 have been shown to function in multiplepathways that involve DNA synthesis: DNA replication acrossG-quadruplex sequence motifs (23–26); break-induced repli-cation (32, 33); and processing of long flaps during Okazakifragment maturation (27–31). Also, in vitro Pif1 increasesstrand-displacement DNA synthesis by Pol δ and allows DNAreplication across arrays of proteins tightly bound to DNA (48).Similarly, here we showed that Pfh1 also stimulates strand-displacement DNA synthesis of Pol δ. Conversely, Pol δ activ-ity stimulates the apparent rate and extent of DNA unwinding.Taken together, these observations suggest that DNA synthesison the 3′-strand prevents the helicase from slipping back on thesubstrate, effectively working as an intrinsic trap in cis thatsuppresses DNA closure. That is to say, DNA synthesis on thenontranslocating strand rectifies the unwinding activity. Wepropose that efficient and processive opening of short stretchesof DNA, the dominant mode of unwinding observed in vitro, isall that the Pif1 family of 5′-3′ helicases needs to achieve whentheir activity is coupled to DNA replication.

MethodsSingle-molecule experiments were performed with an objective-type totalinternal reflection fluorescence (TIRF) microscope (Olympus IX71) with an oil-immersed, high-numerical-aperture TIRF objective (PlanApo N, 60×/1.45N.A., Olympus) and exciting Cy3 with a 532-nm laser (CrystaLaser), as de-scribed previously (65). Cy3 and Cy5 emission intensities were split andrecorded with an Andor iXon EMCCD camera (Model DU897E). The tem-perature of the slide was maintained at 25 °C using a temperature-controlled stage (BC-110 Bionomic controller; 20/20 Technology) and anobjective heater (Bioptechs). A single-channel flow chamber was assembledfrom a coverslip (VWR, 24 × 50 mm N.1) and a predrilled slide (VWR, 75 ×25 × 1 mm). The channel was coated with a solution containing a 1:20 ratio(wt/vol) of biotin-polyethylene glycol (PEG)-succinimidyl valeric acid (SVA)(MWavg 5000) and (wt/vol) mPEG-SVA (MWavg 5000) (Laysan Bio, Arab, AL) in0.1 M NaHCO3 (pH 8.1) and incubated overnight in the dark at 4 °C. The flowchannels were then washed with water, dried with N2, and stored under vacuumconditions. Before the experiment, a 0.2-mg/mL solution of NeutrAvidin(Thermo Scientific) was flowed into the channel and incubated for 2 min,and excess NeutrAvidin was washed away by 200 μL of binding buffer(20 mM Tris·HCl, pH 8.0, 20 mM NaCl, 2% [vol/vol] glycerol, 8 mM MgAc2,1 mM dithiothreitol, 0.1 mg/mL bovine serum albumin). Next, 10 to 100 pMof biotinylated DNA substrate in binding buffer was added to the chamberfor 5 min, free DNA was washed away with 400 μL of binding buffer, andthe DNA on the surface was imaged in imaging buffer (binding buffersupplemented with 0.5% wt/vol glucose, 3 mM Trolox, 165 U/mL glucoseoxidase, and 2,170 U/mL catalase). Next, 100 nM protein was incubated inbinding buffer with the surface-immobilized DNA for 10 min, and excessunbound protein was washed away with 100 to 200 μL of imaging buffer(“wash” condition). Finally, the reaction was started by addition of the indicatedconcentration of ATP in imaging buffer, and images recorded at 30 Hz. Datawere collected using SINGLE (provided by T. J. HA, Johns Hopkins University),processed with IDL (Exelis VIS) and analyzed with MatLab (Mathworks).

Experiments with the protein on the surface were performed by immo-bilizing His6-tagged Pfh1 with a biotinylated PentaHis antibody (45). DNAunwinding experiments monitoring disappearance of the signal from Cy3-labeled DNA substrates, immobilized on a surface, were performed addingat the same time 1 mM ATP (or 1 mM ATP and 100 μM dNTP) and helicase (5to 10 nM) in the absence or the presence of DNA polymerase (20 nM).

Further details on protein purification, DNA substrates, and ensembleassays are provided in SI Appendix.

ACKNOWLEDGMENTS. We thank Timothy Lohman and Tomasz Heyduk forsuggestions and discussions. This work was supported by National Institutesof Health Grants 2R01GM098509 (to R.G.) and 1R01AG062837 (to A.S.).

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