Single-Molecule FRET and Linear Dichroism Studies of DNA Breathing and Helicase Binding at Replication Fork Junctions

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

Carey Phelpsa,b,c, Wonbae Leea,b,c, Davis Joseb,c, Peter H. von Hippelb,c,1, and Andrew H. Marcusa,b,c,1

aOregon Center for Optics, bDepartment of Chemistry and Biochemistry, and cInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403

Contributed by Peter H. von Hippel, August 19, 2013 (sent for review May 29, 2013) DNA “breathing” is a thermally driven process in which base- of adjacent bases to lift off of one another without breaking paired DNA sequences transiently adopt local conformations that interbase hydrogen bonds. Such fluctuations could be involved depart from their most stable structures. Polymerases and other in transition states leading to intercalation and base “flipping.” proteins of genome expression require access to single-stranded More-complex breathing fluctuations could be described as DNA coding templates located in the double-stranded DNA “inte- combinations of the above (or other) breathing modes. Recently, rior,” and it is likely that fluctuations of the sugar–phosphate back- equilibrium and steady-state spectroscopic approaches have been bones of dsDNA that result in mechanistically useful local base pair used to monitor position-dependent DNA breathing that occurs opening reactions can be exploited by such DNA regulatory pro- in the proximity of ss–dsDNA forks and primer–template (p/t) teins. Such motions are difficult to observe in bulk measurements, junctions of DNA constructs (5, 6). Such motions are believed to both because they are infrequent and because they often occur on occur in the microsecond range (7), a difficult regime to access microsecond time scales that are not easy to access experimen- experimentally. Methods to observe such breathing motions of tally. We report single-molecule fluorescence experiments with dsDNA backbones directly, or to determine how these motions polarized light, in which tens-of-microseconds rotational motions might relate to the concerted and biologically relevant activities of of internally labeled iCy3/iCy5 donor–acceptor Förster resonance nucleic acid enzymes, have been largely unavailable. fl energy transfer uorophore pairs that have been rigidly inserted In this paper, we use single-molecule (sm) methods to ex- BIOPHYSICS AND into the backbones of replication fork constructs are simulta- amine DNA breathing and its role in the binding and assembly COMPUTATIONAL BIOLOGY neously detected using single-molecule Förster resonance energy mechanisms of the bacteriophage T4 replication (primosome) transfer and single-molecule fluorescence-detected linear dichro- helicase. Replication helicases are ATP-dependent molecular ism signals. Our results reveal significant local motions in the motors that unwind dsDNA at replication forks to expose tem- ∼100-μs range, a reasonable time scale for DNA breathing fluctu- plates for DNA synthesis (8, 9). The T4 replication system is an ations of potential relevance for DNA–protein interactions. More- excellent model for studies of replication in higher organisms, be- over, we show that both the magnitudes and the relaxation times cause it is the simplest system to use a hexameric helicase–primase of these backbone breathing fluctuations are significantly per- (primosome) subassembly to unwind the base pairs ahead of the turbed by interactions of the fork construct with a nonprocessive, polymerases at the replication fork and to catalyze the synthesis weakly binding bacteriophage T4-coded helicase hexamer initia- of the RNA primers required for the reinitiation of lagging strand synthesis at the 3′ ends of the newly formed Okazaki frag- tion complex, suggesting that these motions may play a funda- – mental role in the initial binding, assembly, and function of the ments (10 12). processive helicase–primase (primosome) component of the bacte- In order for the gp41 helicase to recognize and interact with the replication fork, it is necessary that the bases near the fork riophage T4-coded DNA replication complex. fi smFRET | single-molecule linear dichroism | thermal fluctuations | Signi cance T4 primosome helicase | DNA helicase Unique single-molecule fluorescence techniques were used to “ ” NA “breathing” is defined as the transient opening, due to monitor DNA breathing at and near the junctions of model Dthermal fluctuations, of nucleic acid base pairs at experi- DNA replication forks on biologically relevant microsecond-to- mental temperatures below the melting temperature of dsDNA millisecond time scales. Experiments performed in the absence duplex. Such fluctuations are thought to be important to the and presence of helicase complexes addressed the role of these fl function of replication, transcription, recombination, and repair uctuations in helicase function during DNA replication. These systems, which depend on the ability of the relevant protein studies simultaneously monitored single-molecule Förster res- fl complexes to gain access to ssDNA templates that are located onance energy transfer and single-molecule uorescence linear “ ” in the dsDNA “interior” (1–3). Furthermore, the ability of the dichroism of internal Cy3/Cy5 labels placed rigidly into the genome to spontaneously expose partial template sequences is DNA backbones at positions near the fork junction. Our results fi a central feature of protein–DNA binding models in which open showed signi cant breathing at the fork junction that was conformations serve as substrates that can be “trapped” by a greatly augmented by the presence of weakly bound helicase, fl functional protein complex. McConnell and von Hippel (4) sug- followed by still larger uctuations and strand separation after gested that breathing of duplex DNA might be structurally full duplex DNA unwinding by the complete tightly bound and decomposed into a set of distinct breathing elements, each in- processive helicase complex. volving characteristic fluctuations of the sugar–phosphate back- Author contributions: C.P., P.H.v.H., and A.H.M. designed research; C.P., W.L., and D.J. bone and the nucleobases. For example, a possible motion might performed research; D.J. contributed new reagents/analytic tools; C.P., W.L., D.J., and A.H.M. involve compression along the double-helix axis (perhaps coupled analyzed data; and C.P., P.H.v.H., and A.H.M. wrote the paper. – with twisting or bending), leading to the disruption of Watson The authors declare no conflict of interest. Crick hydrogen bonds without base unstacking. Such motions 1To whom correspondence may be addressed. E-mail: [email protected] or might serve as transition states for low-temperature hydrogen [email protected]. fl exchange. Another class of uctuations might result in extension This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the DNA duplex along its double-helical axis, causing the faces 1073/pnas.1314862110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1314862110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 junction be transiently exposed from behind the sugar–phosphate s A B 1 MHz s / p backbones that shield them from the solvent environment. As phase-modulation shown in Fig. 1 A–C, the T4 gp41 helicase hexamer binds weakly p to the replication fork junction, and is more likely to dissociate

from the fork rather than to engage in processive dsDNA un- Biotin 3’ 5’ PEG z winding (13, 14). In contrast, the T4 primosome helicase, which prism x consists of a gp41 hexamer and a gp61 monomer, binds tightly Neutravidin at the replication fork junction and manifests very processive y unwinding activity in the presence of NTP. The breathing fluc- sample x-y tuations of base pairs located in the immediate vicinity of a objective scanning stage replication fork or a p/t junction are much more extensive than Cy5 those of base pairs located deep within a dsDNA region (6, 13). long-pass iCy5 A hypothetical free-energy surface (FES) describing base-pair iCy3 pinhole filter dynamics near the fork junction is portrayed in Fig. 1D. In this fl dichroic model, base pairs can thermally uctuate into the open state, GTP which is separated from the native DNA structure by a free- 10-mer (ds) 16-mer (ds) mirror phase energy barrier. The height of this barrier is likely dependent on synchronous several variables, such as salt concentration, pH, base composi- photon-counting fl tion, and base pair sequence. By observing the uctuations of detectors dsDNA constructs within regions that have been labeled with fluorescent dyes, it should be possible to obtain information Lagging strand Leading strand C about the FESs to which defined segments of DNA bases and sugar–phosphate backbones are subject, and also to deter- 34-mer (ss) mine how these surfaces are affected by the presence of the ^x T4 helicase. GTP ^y We here perform single-molecule experiments on DNA repli- 29 dT cation fork constructs that have been “internally” labeled with the ^ – z Förster resonance energy transfer (FRET) donor acceptor chro- T4 helicase 3’5’ mophores iCy3 and iCy5 (14). These cyanine dyes were rigidly incorporated into the sugar–phosphate backbone using phos- Fig. 2. The smFRET and smFLD experimental layout. (A) The T4 gp41 heli- phoramidite chemistry, with each fluorophore replacing an op- case binds to the d(T)29 loading sequence on the lagging strand of a model ∙ ∙ posed DNA base at each label position (Fig. 2A). In some DNA replication fork construct. An assembled (gp41)6 gp61 DNA primosome experiments, the fluorophores were positioned close to the rep- complex can unwind the duplex region of the DNA in the presence of GTP. lication fork junction; in others, deep within the DNA duplex. Our The strands within the dsDNA region are internally labeled with the FRET measurements use a polarized excitation scheme that simulta- donor–acceptor chromophores iCy3 and iCy5, respectively. (B) TIRF excitation neously monitors, on submillisecond time scales, the smFRET scheme and detection method. The polarization of the excitation beam is between the iCy3/iCy5 donor–acceptor chromophore pair and the modulated at 1 MHz. The p-polarization component points in the direction fl of the y axis, and the s-polarization component is contained within the x–z uorescence-detected linear dichroism (smFLD) of the iCy3 do- plane. (C) Orthogonally polarized directions used to measure the FLD signal, nor chromophore. The principle of these measurements is based from the perspective of the incident beam. on the idea that a single-oriented DNA replication fork construct will absorb light with different probabilities, depending on the polarization of the laser field. By rapidly modulating the laser signal is sensitive to the relative separation and orientation of the polarization, and recording the phase of the modulation for iCy3/iCy5 chromophore labels, the smFLD signal is sensitive to the every detected signal photon, we were able to monitor the time- iCy3 orientation within the laboratory frame. DNA breathing and dependent projections of the absorption transition dipole moment fl fi helicase-facilitated duplex unwinding are expected to in uence both onto arbitrarily de ned laboratory axes. Whereas the smFRET types of signals. Single-molecule FRET experiments have previously been used to examine helicase binding at replication fork junctions (15). Moreover, polarization-resolved single-molecule experiments have DNA replication fork A lagging strand B 3’ previously been used to study the orientations of chromophores leading strand embedded in complex systems, including glasses (16, 17), bio- 5’ DNA·(gp41) 6 + logical macromolecules (18–20), conjugated polymers (21), and (gp41) helicase 6 light-harvesting complexes (22). The majority of these methods D are capable of sampling a single-molecule signal with time res- gp61 primase ’closed’ fork olution on the order of tens of milliseconds or longer. To observe conformation DNA breathing, it was necessary to record single-molecule signals with submillisecond time resolution.

C free energy lagging strand ‘open’ fork conformation smFLD and smFRET leading strand

DNA·(gp41)6·gp61 local conformational coordinate Q (e.g. inter-base separation, orientation) We performed control experiments on a model DNA replication fork construct that had been internally labeled using the donor– Fig. 1. (A) The isolated hexameric gp41 helicase (blue spheres), the subunits acceptor FRET chromophore pair iCy3/iCy5 as shown in Fig. 2A. of which are bound together by intersubunit (and in this case non- In this way, both smFLD and smFRET signals could be simul- – hydrolyzable) NTP ligands (not shown for clarity), binds weakly to the ss taneously monitored to provide information about DNA back- dsDNA junction of a replication fork, favoring dissociation over binding, as bone rigidity and the relative displacement of the conjugate strands shown in B. Introduction of gp61 primase (orange ellipsoid), in a 6:1 gp41-to- gp61 subunit ratio, causes the resulting T4 primosome helicase complex to at the level of the Cy3/Cy5 probes. Ensemble linear dichroism (LD) bind strongly to the replication fork, as shown in C.(D) A hypothetical FES measurements are often used to determine the orientations of ab- describing local dsDNA ﬂuctuations near the fork junction in the absence sorption transition dipole moments of chromophores embedded in of helicase. uniaxially strained polymer ﬁlms (23, 24). The LD of such samples

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1314862110 Phelps et al. Downloaded by guest on September 25, 2021 is defined as the difference between the absorption of light polar- of photoelectron counts vs. polarization phase ϕ approximates ðϕÞ = + ðϕ + ϕ Þ ized parallel and perpendicular to the strain axis. a smoothly varying sinusoidal function f A C cos 0 , where 2C is the modulation amplitude (equal to the FLD signal), = − [1] ϕ LD Ak A⊥ A is a constant offset, and 0 is a known phase determined by the modulation electronics. We numerically calculated the smFLD ðϕÞ ðϕ + ϕ Þ In FLD, the relative population of molecules excited in the signal by multiplying the function f by cos ref , where we ϕ = ϕ two polarization directions is determined by measuring the dif- set ref 0, and subsequently performing an average over the ference between the corresponding fluorescence intensities. In phase angle ϕ: our smFLD experiments, the DNA replication fork substrates D E were chemically attached to the surface of a glass microscope C FLD cos ϕ + ϕ fðϕÞ = = : [2] slide using biotin/NeutrAvidin linkages (Fig. 2A and Sample ref ϕ 2 4 Preparation Methods). The DNA fork construct, with its rigidly attached iCy3 donor chromophore, was thus oriented with re- We define the reduced FLD (FLDr) = FLD/Nhist, where Nhist spect to the laboratory frame. A total internal reflection fluo- is the number of photoelectron counts contributing to the in- rescence (TIRF) microscope was used to illuminate the sample tegrated histogram. The quantity FLDr is independent of signal with an evanescent field, as shown in Fig. 2B. The polarization of intensity, and thus depends only on the orientation of the mo- the incident laser beam was rapidly modulated (at 1 MHz) so lecular transition dipole moment. We found that accurate results that the sample was alternately excited using plane polarizations could be obtained with as few counts as Nhist = 20. Thus, a in the directions –45° (designated as k) and +45° (designated smFLD signal trajectory could be obtained from a molecule r − as ⊥) relative to the surface normal (Fig. 2C; see instrument with an emission count rate of ∼25,000 s 1 and a time resolution details provided in SI Text, Instrumentation for Single-Molecule of ∼800 μs. Fluorescence-Detected Linear Dichroism and Single-Molecule För- The second method we used to process our trajectories is ster Resonance Energy Transfer). Because the absorption proba- based on an analysis of time correlation functions (TCFs). bility depends on the square projection of the laser field onto the We focus on the fluctuatingsmFRETandsmFLDr signals, excitation transition dipole moment, the emission probability FRETðtÞ = δFRETðtÞ + FRET and I ðt; ϕÞ = δI ðt; ϕÞ + I ,re- ϕ T T T depends on the phase of the polarization modulation cycle. The spectively, which were recorded using a DNA replication fork modulation amplitude of the fluorescence intensity is a measure of construct at equilibrium. We define the TCFs GFRETðτÞ = the orientation of the absorption transition dipole moment. h ðτÞ ð Þi ðτÞ = h p ðτÞ ð Þi FRET FRET 0 and GFLD IT IT 0 ,whichdescribe The 1-MHz polarization modulation of the excitation beam BIOPHYSICS AND the decay of correlations of the fluctuating smFRET and smFLDr was implemented using acousto-optic Bragg cells placed in the signals, respectively, with increasing time interval τ. The angle COMPUTATIONAL BIOLOGY beam path of the laser (SI Text, Instrumentation for Single-Molecule brackets indicate an average over time according to Fluorescence-Detected Linear Dichroism and Single-Molecule Förster Resonance Energy Transfer). The sample image was raster- Z∞ scanned using a computer-controlled x-y piezo-scanning micro- p G = ðτÞ = SðtÞS ðt + τÞdt: [3] scope stage (NPS-XY-100A; Queensgate), and the stage position FRET FLD was held fixed when the fluorescence from a single molecule was −∞ detected. Donor and acceptor fluorescence from a single mole- 3 ð Þ fl cule was masked using a pinhole, directed along separate paths In Eq. , S t represents the uctuating smFRET or smFLDr using a dichroic beam-splitter, and separately detected using two signal. The TCFs, so defined, are expected to decay on a time fast photon-counting detectors (SPCM-AQR-16, 175-μm active scale associated with the same underlying microscopic processes area; Perkin-Elmer Optoelectronics). The detection times of in- that give rise to spontaneous fluctuations of the system away dividual photoelectron counts were stored on a computer and from its stable equilibrium configuration (26). We note that additionally assigned values of the laser polarization phase ϕ, the short time limit of Gð0Þ = hS2i represents the mean square which was subdivided into 64-bin increments. We thus recorded magnitude of the fluctuating signal, whereas the long time limit time- and ϕ-dependent trajectories of the donor (acceptor) sig- Gð∞Þ = hSi2 represents the square mean signal. The TCF anal- P ð Þ ð Þ ð ; ϕÞ = NDðAÞ δð − D A Þ ð ϕDðAÞÞ D A ysis provides access to time scales much faster than the histogram nals, IDðAÞ t n = 1 t tn exp i n , where tn and ϕDðAÞ are the time and phase of the nth donor (acceptor) signal integration method described previously, because the latter relies n on multiple detection events to reconstruct a sinusoidal wave- count, respectively, and NT = ND + NA is the total number of signal counts. In principle, single-molecule kinetic information form. Because the TCF method correlates the properties of in- could be extracted from these data with a time resolution of ∼1 μs dividual photoelectron signal counts, we were able to extract and a polarization phase resolution of ∼2π/64 ≈ 0.1 rads. From kinetic information from single-molecule trajectories with ∼20-μs the signal trajectories ID=Aðt; ϕÞ, we constructed the cumulative time resolution. We performed control experiments to calibrate the smFLD donor–acceptor signal IT = ID + IA and the FRET efficiency parameter, defined as I =I . We note that the presence of the FRET and smFRET signals from known test samples, and to ensure A T fl acceptor chromophore does not alter the meaning of the FLD that our measurements were not in uenced by the dynamics of measurement because the acceptor signal depends on the excita- laser-induced excited triplet states (SI Text, Sample Preparation tion of the donor chromophore, which was excited with polariza- and Model DNA Replication Fork Constructs). fi ϕ tion speci ed by . Two separate approaches were taken to Results process these data. The first approach was used to visualize the smFLD and smFRET trajectories on millisecond time-scales, We investigated the dynamics of the two iCy3/iCy5-labeled DNA and the second served to resolve the tens-of-microseconds dy- replication fork constructs (SI Text, Sample Preparation and namics of the DNA sugar–phosphate backbone. Model DNA Replication Fork Constructs). For both types of DNA Because the fluorescence intensity from single iCy3/iCy5- constructs, the iCy3/iCy5 chromophores replaced nucleotide − labeled molecules was of the order of ∼25,000 s 1, we integrated bases on opposite strands directly across from one another. “ ” the cumulative signal IT over ∼800 modulation cycles to obtain The duplex-labeled construct had the iCy3/iCy5 pair placed a ϕ-dependent probability distribution, from which the smFLD deep in the dsDNA region of the fork construct, whereas the signal could be determined. We extracted the smFLD signal “fork-labeled” construct contained the iCy3/iCy5-labeled probe through a post data-acquisition procedure that was operationally pair at the ss–dsDNA junction. Using these DNA constructs, we similar to the algorithm used by a lock-in amplifier (25). For were able to simultaneously measure the smFRET and smFLDr a single molecule of fixed orientation, the integrated histogram signals. We initially examined the duplex-labeled DNA construct

Phelps et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 and its unwinding by the T4 primosome helicase complex. In the placed at either the ss–dsDNA fork junction or deep within the absence of any helicase, we observed that smFRET signals were duplex region. Our initial studies focused on observing dynamics constant over time, with an average FRET efficiency IA=IT ≅ 0.7. revealed by calculating two-point TCFs for the smFRET and Photobleaching from these samples occurred only occasionally, smFLDr trajectories, with the goal of observing DNA breathing presumably because the fluorophores were protected from the fluctuations at equilibrium. The TCFs should reveal thermally solvent environment by the sugar–phosphate backbones of the activated backbone fluctuations as the iCy3/iCy5 probes sample fi dsDNA (14). In the absence of unwinding proteins, the smFLDr the FES shown hypothetically in the simpli ed diagram of Fig. signals also remained at a constant value, which could lie anywhere 1C. The decays of the TCF report on the timescale of these within the range −1 to 1. Example control data sets for duplex- equilibrium fluctuations, and the functional form of the decay, labeled constructs are presented in SI Text, Control Experiments. can provide information about the nature of the fluctuations. Upon introducing the T4 hexameric helicase and 6 μM ATP In Fig. 4, we show examples of TCFs calculated from simul- into the sample chamber, DNA-unwinding events could be ob- taneously recorded smFRET and smFLDr trajectories. Though served that were characterized by a sudden drop in the iCy5 the paired TCFs derived from smFRET and smFLDr signal traces ∼ μ fluorescence intensity and a simultaneous increase in the iCy3 decayed on time scales of the order of 100 s, they generally fl fluorescence intensity, defined as a drop in the FRET efficiency. appeared to be nonidentical, re ecting the distinct origins of the Similar helicase-unwinding FRET-conversion events have been two types of signals. The decays of the TCFs should be sensitive to the motions of the rigidly integrated probes, and thus of the DNA observed previously with this system (14). smFLDr traces were fi – sugar–phosphate backbones, and hence likely reflect important also signi cantly affected by these events. In Fig. 3 A C,we “fl ” present simultaneously measured single-molecule iCy3/iCy5 in- aspects of DNA breathing. To ensure that potential ickering dynamics of long-lived excited triplet states did not significantly tensities, representing smFRET and smFLDr trajectories, respectively, of the duplex-labeled DNA replication fork construct affect our results (27), we performed control studies to check that in the presence of the processively unwinding T4 helicase–pri- the observed TCFs were independent of the excitation intensity of mase system (300 nM gp41 and 50 nM gp61) and 6 μM ATP. the laser (SI Text, Control Experiments). In Fig. 4, we show results Before the unwinding event at the ∼17.5-s tick mark, the iCy5 from the fork-labeled DNA construct in the absence of helicase proteins. Additional examples of data sets are presented in SI Text, intensity remained high and constant, resulting in a FRET effi- Control Experiments, and show those of the same DNA construct ciency of over 0.7. During this time, the smFLD signal fluctuated r in the presence of gp41 helicase hexamers that had been assem- near a nonzero constant value. Immediately after the unwinding bled using nonhydrolyzable GTPγS as the NTP ligand. These event at ∼17.5 s, characterized by a drop in the smFRET signal, fl helicase hexamers, assembled with nonhydrolyzable NTPs in the the smFLDr signal uctuated broadly around zero because the absence of primase, bind weakly to the ss–dsDNA junction of the iCy3-labeled strand had been freed from the tethered dsDNA replication fork constructs, but cannot catalyze duplex unwinding construct by the helicase. In Fig. 3 Right, we also show selected ϕ (5, 13, 14). polarization -dependent signal distributions, which were used Our experimentally derived TCFs are consistent with earlier to determine the smFLDr trajectory shown in Fig. 3B. reports from ensemble fluorescence correlation spectroscopy Having established that our experiment was able to simulta- (FCS) experiments performed by Altan-Bonnet et al. (7), in neously monitor the smFRET and smFLDr trajectories, we next which DNA breathing dynamics were observed in the 30- to 100-μs sought to observe the breathing dynamics of the system using range. These authors proposed a model to explain the functional model replication fork constructs with the iCy3/iCy5 probes form of the TCFs measured in their solution phase experiments that was based on a rate equation in which a DNA breathing “bubble” can open or close by 1 bp, with the closing rate much faster than the rate of opening. The TCFs produced using this A 600 1.0 Cy3 0.8 model have a functional form 500 Cy5 400 0.6 rffiffiffiffiffiffiffi rffiffiffiffiffiffiffi τ τ τ −τ= τ 300 0.4 GðτÞ ∝ 1 + erfc − e 4 c : [4] 0.2 τ τ πτ No. Counts 200 2 c 4 c c integrated signal 100 0.0 0 102030405060 4 fi B 1.0 1.0 We have used Eq. to t the TCFs calculated from our single- 0.8 molecule experiments, including those shown in Fig. 4, and in r 0.5 fi 0.6 many cases we found that this model t our data very well. Ap- FLD 0.0 proximately 50% of the molecules we observed in the absence of 0.4 fi ∼ -0.5 0.2 unwinding proteins t this model precisely, whereas 25% of the integrated signal 0.0 molecules we observed in the presence of unwinding proteins also 0 102030405060 fi C 0.8 t the model. For those molecules in which there is a discrepancy, 1.0 the model generally does not match the fastest time components 0.6 0.8 of the decays, and therefore a refinement to the model may be ‘duplex-labeled’ 0.6

FRET 0.4 fork construct necessary to capture the shortest time dynamics to which our 0.4 experiments are sensitive. Because the closing rate of the DNA 0.2 0.2

integrated signal bubble should be faster than the opening rate, the relaxation 0.0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 times reported in ref. 7 and in our data can be considered to time (sec) represent the lifetime of the open state of the bubble. The moderate agreement we observe between this model and our Fig. 3. (A) Single-molecule iCy3/iCy5 signals were recorded during a T4 pri- – experimental data suggests the possibility that the chromophore- mosome helicase unwinding experiment. An unwinding event occurred at the labeled region of the DNA construct may experience multiple 17.5-s tick mark. (B) The simultaneously recorded smFLDr signal exhibits a change in behavior coincident with the unwinding event defined by the bubbles of various sizes over the course of the observation time fi window. We note that the average lifetime we observed in our smFRET conversion ef ciency shown in C. Horizontal dashed lines (red/green) ∼ – indicate the magnitude of the fluctuations immediately before and after the system is 4 5 times larger than that observed in ref. 7, which is unwinding event. (Right) Laser polarization ϕ-dependent signal distributions likely due to the different labeling schemes used in the two

that were used to determine the FLDr signal shown in B. The donor–acceptor studies. The internally labeled iCy3 and iCy5 chromophores re- chromophore labels are placed deep within the double-stranded region of the place native bases in our DNA strands, and this relatively rigid DNA replication fork construct (SI Text, Sample Preparation and Model DNA conﬁguration might extend the lifetime of spontaneously formed Replication Fork Constructs). bubbles in the local dsDNA sequence surrounding the probes. In

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1314862110 Phelps et al. Downloaded by guest on September 25, 2021 SM FRET TCF SM FLD TCF fork construct (red, 289 μs). The faster decay time for the duplex ABr sample suggests that the closing rate of a spontaneously formed = 8.9 × 10-3 5 = 6.1 × 10-2 bubble in the duplex region of dsDNA is faster than that of

2.8 ) -2 2 -3 a similarly formed unstable conformation near the ss–dsDNA

) = 2.1 × 10 = 9.8 × 10 2 fork junction. DNA breathing ﬂuctuations have been shown to ‘fork- = c occur much more frequently at replication fork junctions than at labeled’ 516 sec ( ) (×10 ( ) (×10 construct FLD sequences positioned well within duplex DNA regions (5, 6). The G

FRET relative magnitudes of the lifetimes we have observed for the 1 G c = 143 sec fork- and duplex-labeled DNA constructs are consistent with 2.0 previous observations (7). 10-5 10-4 10-3 10-2 10-5 10-4 10-3 10-2 (sec) (sec) The relaxation times of equilibrium fluctuations observed in our data can be dramatically lengthened by the introduction of Fig. 4. Examples of single-molecule FRET and FLDr TCFs. TCFs determined the GTPγS-stabilized hexameric gp41 helicase, which binds weakly from (A) FRET and (B) FLDr trajectories obtained from a single DNA model to replication fork junctions. In this experiment we assembled the fork construct in the absence of helicase proteins. Red lines represent opti- μ mized fits to Eq. 4, where the fit parameter hSi2 is the mean square mag- gp41 helicase (300 nM gp41, no gp61) using 6 M nonhydrolyzable 2 GTPγS rather than NTP. Under these conditions, the hexameric nitude of the signal fluctuations, hS i is square average signal, and τc is the correlation time. The donor–acceptor chromophore labels are placed at the helicase forms and binds weakly at the fork junction, as depicted ss–ds fork junction of the DNA construct (SI Text, Sample Preparation and in Fig. 1 A–C, but unwinds the dsDNA sequence by only one base Model DNA Replication Fork Constructs). pair (13, 14). As shown in Fig. 5B, we observed that the distribution of relaxation times for the DNA construct with chromophore labels placed near the replication fork junction were contrast, the chromophores used in ref. 7 were attached using dramatically broadened (blue), with an average decay time of fl exible linkers outside of the DNA, leaving native bases intact, so 650 μs. Clearly, the presence of the gp41 helicase at the iCy3/ that the dynamics observed are likely perturbed by the enhanced iCy5-labeled replication fork junction extended the lifetimes of mobility of the probe labels. open (unpaired) DNA bubbles formed in that region, and To examine the effects of the presence and initial binding of broadened their distribution. Furthermore, both the width and the helicase protein on DNA breathing, we defined the re- fl τ the average magnitude of uctuations at the replication fork

laxation time as the time required for the TCF to decay to BIOPHYSICS AND c increased in the presence of the T4 helicase. In Fig. 5C,we a factor 1/e of its initial value. In Fig. 5A, we show histograms of

fl COMPUTATIONAL BIOLOGY τ plot the distribution of the magnitude of the relative uctuations the relaxation times ( c) obtained from the TCFs of the smFRET h 2i − h i2 data using DNA fork constructs that had been labeled either of the smFLDr signal S S ,whichcanbeusedtocharacterize deep in the duplex region (blue) or at the replication fork the width of the distribution of open DNA fork conformations. junction (red). We characterized these data using the gamma Though both the duplex- and fork-labeled DNA constructs exhibit α −1 α−1 −τ =β distribution function fðτ Þ = ½β ΓðαÞ ðτ Þ e c , which is an a relatively narrow distribution of open conformations (Fig. 5C, c c fl appropriate distribution for the time domain. Here, the param- Left and Center), the average magnitude and width of the uc- eters α and β describe the degree of skewednessR and the width tuations at the replication fork junction increases in the pres- Γð Þ = ∞ z−1 −t ence of the T4 helicase (Fig. 5C, Right). The combination of of the distribution, respectively, and z 0 t e dt is the gamma function. We also present the corresponding rate domain these results for the helicase-dependent distributions of relaxation fl (kc = 1=τc) histograms, which are fit to Gaussian distributions in times and uctuation magnitudes is consistent with the notion that SI Text, Control Experiments. In the absence of unwinding pro- microsecond time-scale breathing fluctuations at and near the fork teins (Fig. 5A), the duplex-labeled construct exhibits a slightly junction likely play a significant role in helicase-binding mecha- faster average decay time (blue, 201 μs) in comparison with the nisms at this locus.

Fig. 5. Dynamics of breathing ﬂuctuations at the replication fork in the absence and presence of helicase. (A) Histograms of relaxation times (τc) obtained from the analysis of smFRET/smFLD trajectories for DNA fork constructs, which were labeled with iCy3/iCy5 placed deep in a duplex region (blue) or at the replication fork junction (red) in the absence of helicase protein. (B) A comparison is shown for a fork-labeled construct in the absence (red) and C the presence (blue) of the “frozen” hexameric helicase (gp41 ∙ GTPγS)6 composed of 300 nM gp41 and 6 μMGTPγS. (C) A comparison is shown of histograms of the relative magnitudes hS2i − hSi2 of the

ﬂuctuating smFLDr signal for duplex-labeled DNA (Left), fork-labeled DNA (Center), and fork-labeled

DNA + (gp41 ∙ GTPγS)6 (Right). Histograms of the relaxation times were characterized using the gamma distribution function, given in the text, with skewed- ness and width parameters α (dimensionless) and β (in units of milliseconds), respectively.

Phelps et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 Discussion influence one another, in which case the formation of the primo- Observations of the local conformations of macromolecules some–DNA complex must reflect a random coincidence of events. through smFRET and smFLD should provide insights into the Though the latter scenario can describe the chemical kinetics of functional mechanisms of the “protein machines” that manipu- small molecular systems, which may be characterized by a station- late DNA. As a helicase processively unwinds a DNA duplex, the ary potential energy landscape, our current results appear to sup- local conformation and flexibility of the opposing DNA strands port the more complex former situation, in which a highly mutable fl must change considerably. Such motions will be re ected by time-evolving energy landscape can respond to locally changing fluctuations in the orientation and separation of fluorophores that – environments, including here the presence of the weakly bound substitute for nucleotides within the sugar phosphate backbone, helicase complex at the DNA replication fork junction. and these motions can be simultaneously monitored using smFLD and smFRET. Methods The smFLD method is a useful complement to smFRET be- Instrumentation for smFLD and smFRET. cause it can help to avoid misinterpretation of false smFRET A smFRET instrument was designed to signals. For example, if the acceptor fluorophore of a coupled use rapid modulation of the excitation beam. The detection system involved donor–acceptor FRET pair was to undergo a photobleach event custom electronics and analysis to perform single photon-counting phase- (permanent or temporary), there would be a drop in acceptor sensitive techniques. Instrument details and theoretical considerations to intensity coincident with a rise in donor intensity. An acceptor perform these experiments are provided in SI Text, Instrumentation for photobleach event could thus be misinterpreted as a sudden Single-Molecule Fluorescence-Detected Linear Dichroism and Single-Mole- separation between the donor–acceptor pair. This ambiguity can cule Förster Resonance Energy Transfer. be addressed by simultaneously monitoring the smFLD signal, because a FRET conversion event due to donor–acceptor pair Sample Preparation and Model DNA Replication Fork Constructs. Model DNA separation will be accompanied by a significant change in the replication fork constructs, which were labeled with the iCy3/iCy5 FRET smFLD signal, whereas an acceptor photobleach event will not. chromophore pairs, were purchased from Integrated DNA Technologies. Our finding that the presence of the gp41 hexameric helicase Microfluidic sample chambers were constructed from microscope slides and greatly enhances the widths and average values of both the life- coverslips. Details of the sample chamber construction, cleaning procedures, time and the magnitude distributions of “open” conformations at and reagents used are given in SI Text, Sample Preparation and Model DNA the fork junction implies that these thermally populated minority Replication Fork Constructs. states of the DNA substrate play a role in the helicase-binding mechanism. The significance of our results lies in the context of ACKNOWLEDGMENTS. The authors thank Shamir A. Kansakar, who partic- understanding the energetic factors that contribute to the func- ipated in the Summer Program for Undergraduate Research at the University tional assembly of biological macromolecular machines. An open of Oregon during the summer of 2012, for his assistance in the preparation – of samples and sample cells used in our smFLD and smFRET experiments; question is whether the initial steps of helicase primase (primo- Clifford Dax for his technical assistance in developing the custom digital some) assembly occur at the replication fork junction through electronics used in our smFRET/smFLD detection system; Neil Johnson for a cooperative mechanism in which the binding of the gp41 hex- helpful conversations; and Steve Weitzel for providing the T4 gp41 and gp61 americ helicase fundamentally alters the nature of DNA fluctu- preparations used in these studies. This work was supported by National ations at the fork junction and thereby facilitates the subsequent Institutes of Health/National Institute of General Medical Sciences Grant GM-15792 (to P.H.v.H.); Office of Naval Research Grant N00014-11-0193 (to binding of the gp61 primase during initial primosome helicase A.H.M.), and National Science Foundation, Chemistry of Life Processes Program assembly, as well as during the subsequent unwinding reaction. Grant CHE-1105272 (to A.H.M.). P.H.v.H. is an American Cancer Society Research Alternatively, DNA breathing and helicase binding might not Professor of Chemistry.

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