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Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA

In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its functions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA unwinding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 5′ to 3′ directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 5′ to 3′ directionality.

The stoichiometry of Pif1-DNA complexes and their sedimentation coefficient can be extracted from sedimentation velocity profiles of labeled oligonucleotides in the presence of different concentrations of Pif1 helicase, taking advantage of the large difference in sedimentation coefficient/molecular weight of free vs bound ssDNA (84). Suppose a given amount of labeled ssDNA is sedimented in the presence of a given amount of Pif1 helicase and only the signal from the ssDNA is monitored. At the beginning of a sedimentation velocity scan the higher molecular weight complexes of ssDNA-protein, which have higher sedimentation coefficient, have not yet sedimented. The observed value of absorbance represents the total concentration of the input ssDNA. As sedimentation scans proceed the high molecular weight species will start sedimenting and over time a plateau will be established. The absorbance of this newly established plateau represents the concentration of the free ssDNA which sediments slower that the ssDNA-protein complexes. The difference between the new plateau and the one determined at the  Figure S1d shows the c(s) distribution as a function of s 20,w calculated from the s2 velocity profiles in Figure S1a-c. For reference the c(s) distribution determined under the same solution conditions for a monomeric Pif1 is show in grey (23). Independently of the DNA:Pif1 loading ratio the fast moving boundary in Figures S1a-c shows one peak that sediments slightly faster than the monomer of Pif1 ( Figure S1d, black traces). This is due to the finite contribution of the ssDNA to the sedimentation coefficient of the Pif1-DNA complex, indeed for shorter ssDNA the observed sedimentation coefficient of the complex shifts towards the value of free Pfi1 (data not shown). The observed sedimentation coefficient strongly suggests that in the protein-DNA complex formed in excess of DNA, Pif1 is a monomer. Only in the presence of excess Pif1 relative to ssDNA the sedimentation coefficient is dramatically increased, suggesting the formation of at least a dimer of Pif1 on DNA (Fig S1c, dotted gray).
Finally, in presence of excess DNA a second peak is also observed at smaller s 20,w values. This corresponds to the free ssDNA and its contribution to the c(s) increases as the DNA:Pif1 ratio increases, due to the increase in the concentration of free ssDNA.
The data in Figure S1 clearly show that when a complex of Pif1 with ssDNA is formed in the presence of a 2:1 ratio of DNA to protein, as is the case for the translocation experiments presented in the main text, a single monomer of Pif1 is bound to the nucleic acid.

ATPase activity is required for Pif1 translocation.
In Figure 1b we used the non-hydrolizable ATP analog ATPγS to conclude that ATP hydrolysis drive Pfi1 translocation on ssDNA. Additional control experiments to support this conclusion are shown in Figure   S2. Figure S2a shows the same experiments as in Figure 1b but performed either with AMP-PNP, a different ATP analog, or with ATP but in the presence of added 7mM EDTA to Buffer T 200 (containing 5mM MgCl 2 ). As observed for the experiments with ATPγS (gray trace), when a preformed Pif1-ssDNA complex is mixed with 1mM AMP-PNP and 0.1 mg/mL heparin (red trace) Pif1 dissociates from ssDNA.
Interestingly the apparent rate of dissociation in this case is different from the one observed with ATPγS, suggesting that although both nucleotides are ATP analogs they might bind with different affinity to Pif1 or affect differently its binding to DNA. Dissociation of Pif1 is also observed when a preformed Pif1-ssDNA complex is mixed with 1mM ATP and 0.1 mg/mL heparin in the presence of added 7mM EDTA, consistent with the need of ATP hydrolysis to drive translocation.
Finally, we generated the ATPase "dead" version of Pif1 to show that it does not support unwinding (23).
The comparison of the translocation activity between Pif1 and its catalytic "dead" variant is shown in Figure S2b. For Pif1 K264A no effect on the Cy3 fluorescence after mixing with ATP is observed, supporting the conclusion that Pif1 ATPase activity is required to drive translocation.
The presence of a slow moving peak is not due to either the location of the fluorophore, an effect of heparin or an altered ATP binding site.
The data in Figure 2 and 3a in the main text clearly show that at the lower NaCl concentration translocation of Pif1 on ssDNA is accompanied by the appearance of a second peak in the Cy3 signal. Of s3 all the fluorophores tested (carboxyfluorescein, fluorescein, AlexaFluor488, AlexaFluor546, TYE563, TAMRA) we found that only Cy3 provides sufficient and reliable signal to monitor translocation. To test whether the appearance of second peak is strictly due to a fluorophore dependent effect we employed ssDNA substrates where the location of Cy3 is changed. Whether the Cy3 fluorophore is linked at the 3'end of the ssDNA via a C3 linker or it is placed at an internal position has almost no effect on the translocation behavior of Pif1 ( Figure S3b). This, in conjunction with the observed NaCl concentration and ssDNA length dependences, provides strong evidence that this behavior does not originate exclusively from a fluorophore dependent effect induced by Pif1 binding but rather from Pif1 translocating along ssDNA.
Next, we tested whether the presence of heparin as a trap for Pif1 could contribute to the observed behavior of the Cy3 fluorescence time courses and to the appearance of the unexpected slow moving peak. Figure S3c shows a comparison of the Cy3 fluorescence time courses after mixing in Buffer T100 a pre-formed complex of Pif1 and different lengths of 3'-end labeled ssDNA (dT n -Cy3) with 1mM ATP and either 0.1mg/mL (black) or 0.8 mg/mL (gray) heparin. It is evident that a 8-fold higher concentration of heparin slightly affects the maximum amplitude of the fast moving peak while its position is little affected, suggesting that in this concentration regime heparin has a small effect on the translocation behavior of Pif1. Most importantly, independent of the concentration of heparin, the slow moving peak observed at longer times is still present and within error unaffected. This strongly suggest that the presence of the observed second peak in the Cy3 fluorescence time courses does not originate from a heparin effect on either the translocation behavior of Pif1 or the monitored signal.
One simple possibility for the origin of the observed second peak could be the presence of a small fraction of Pif1 that has altered ATP binding/hydrolysis. However, inclusion in the purification procedure of an ATP-agarose chromatography step (which selects for Pif1-ATP binding) has no effect on the observed translocation signal ( Figure S3d). Moreover, we tested whether the observed second peak originates from accumulation during the cycle of ATP hydrolysis of a slow dissociating ADP-bound intermediate of Pif1 that exchanges for ATP slowly during the time of translocation. If this were the case translocation experiments performed at different ADP/ATP ratios should affect the fraction of the observed second peak. This is not the case. The change in Cy3 fluorescence after mixing a pre-formed complex of Pif1 with dT 38 -Cy3 with 0.2mM ATP in the presence of different concentrations of ADP and 0.1mg/mL heparin in Buffer T100 is shown in Figure S3e. It is evident that Pif1 translocation is not affected by the presence of ADP up to a 1:1 ratio with ATP. Interestingly, translocation starts to be affected in the presence of a 10fold excess of ADP over ATP. However, in this case both observed peaks shift to longer times while the relative fraction remains unaffected. These data strongly suggest that the observed second peak does not originate from accumulation during the cycle of ATP hydrolysis of a slow dissociating ADP-bound s4 The appearance of the second translocating population is not linked to the nature of the base of the NTP hydrolyzed.
Next we tested whether the observed second peak originates from the specificity of Pif1 for ATP binding and hydrolysis. It has been reported that Pif1 hydrolyzes GTP at a much slower rate than ATP (3). Although little is known on the ability of GTP to support the activities of Pif1 we found that GTP is able to sustain Pif1 translocation, albeit at a slower rate as compared to ATP. Figure  In order to test whether the appearance of the observed second peak in Cy3 fluorescence is temperature dependent, we determined Pif1 translocation on the different lengths of ssDNA at three additional temperatures. Consistent with the observation that the second peak in Cy3 fluorescence becomes apparent for the longer ssDNA lengths, at all the temperatures examined Pif1 translocation on a 20-nt long ssDNA is not accompanied by a detectable contribution of the second moving population of Pif1 to the total fluorescence signal ( Figure S4a). The situation is different for the 38-nt ssDNA, where now the presence of a second peak in the Cy3 fluorescence becomes evident at any temperature examined ( Figure S4b). At the same time, it is also evident that the apparent translocation rate (i.e. peak position of the maximum fluorescence) is temperature dependent. As a first order approximation (see text for details) the time of the first peak in Cy3 fluorescence follows a linear dependence on the ssDNA length at all the temperatures examined ( Figure S4c). Also, the apparent translocation rate calculate with this approximation shows a linear dependence upon temperature ( Figure S4d), suggesting an activation energy of ~3.7 kcal/mol. Further interpretation of the meaning of this activation energy will require a full analysis of the translocation traces and de-convolution of the contribution of both populations to the observed temperature dependence. s5 The "fast" and "slow" peaks do not interconvert on the time scale of translocation.
We utilized the sensitivity to the concentration of NaCl of the second peak in Cy3 fluorescence ( Figure   3a) to test whether the "fast" and "slow" peaks can re-equilibrate during the time of translocation (i.e. whether they originate from two populations of Pif1 that can interconvert during translocation). If the fast and slow peaks can re-equilibrate during translocation it should be possible to modulate their relative fraction by NaCl concentration jumps. The change in Cy3 fluorescence after mixing a pre-formed complex of dT 38 -Cy3 and Pif1 in Buffer T 100 (buffer that Pif1 is dialyzed in) with 1 mM ATP and 0.1mg/mL heparin in Buffer T300 (corresponding to a jump from 100 to 200mM NaCl final concentration after equal mixing) is shown in Figure S5a  T100 is shown in Figure S5b. It is evident that Pif1 is able to translocate on the ssDNA containing a mixed sequence proximal to the Cy3 label. This direct comparison shows that the presence of the mixed sequence affects both the fraction of translocating Pif1 (amplitude of the signal) and the apparent rate s6 (~peak position), suggesting that translocation of Pif1 is sensitive to the sequence composition of the ssDNA.

Pif1 is able to translocate on ssDNA containing a biotin at an internal position.
We employed streptavidin displacement assay as proof of principle to test whether translocating Pif1 is able to displace a protein bound to ssDNA. Based on this assay the data in Figure 6 strongly suggests that during translocation Pif1 can displace streptavidin bound to the ssDNA. However, it is clear that the efficiency of streptavidin displacement is relatively low. Therefore, we tested whether this could at least in part be due to the presence of biotin. The change in Cy3 fluorescence after mixing a pre-formed complex of Pif1 and dT 30 T(bio)T 7 -Cy3 or dT 38 -Cy3 with 1mM ATP and 0.1mg/mL heparin in Buffer T100 is shown in Figure S5c. It has been shown that the Cy3 signal change induced by the arrival of a protein in its close proximity is sensitive to the distance between the fluorophore and the approaching enzyme (27,83). Therefore, it is possible that the Cy3 signal observed for the biotynilated ssDNA could arise from Pif1 blocked at the biotin modified thymidine. However, in this case the peak position would be consistent with Pif1 translocating along a ~30-mer and the position of the peak should be shifted towards shorter times (closer to the peak position of the 28-mer in Figure 2c). This is not the case. Finally, the presence of a second peak in the Cy3 signal is still observable even for the biotynilated ssDNA, further supporting the conclusion that this behavior is an intrinsic property of translocating Pif1.

Translocation model and nonlinear least squares analysis (NLLS)
We initially analyzed the translocation time courses obtained at 200mM NaCl and 0.5mM ATP (final) using a simple n-step sequential model (a single population in Scheme 1) (27). In this model the translocase is initially randomly bound i translocation steps away from the 3'-end, with a contact size d nucleotides and with concentration, where n is the maximum number of translocation steps needed for a translocase bound initially at the 5'end to move to the 3'-end of a ssDNA that is L nucleotides long. Upon addition of ATP the translocase moves along the ssDNA with finite processivity with a rate constant, k t , and can dissociate from the ssDNA with rate constant k d . Upon reaching the 3'-end of the ssDNA the translocase dissociates from s7 the ssDNA with rate constant k end . Figure S6a shows the NLLS analysis of the time courses collected at 200mM after mixing with 1mM ATP with Scheme 1 for a single population using equation (  Based on the all the experimental evidence discussed, we wondered whether the appearance of a second peak in the Cy3 fluorescence time courses could be due to the presence of a second population of Pif1 translocating on ssDNA. The data were analyzed with the two-population n-step sequential translocation mechanism in Scheme 1, where we assumed that the observed changes in Cy3 fluorescence originate from two independent populations of translocating Pif1 (p1 and p2). Equation (e2) is derived from Scheme 1 using Laplace transforms. Here P1 and P2 is the product of the fractional contribution of each population of the total translocase concentration and the fluorescence signal associated for each population of Pif1 at the 3'-end. We allowed each Pif1 population to have unique kinetic parameters for translocation (k t p1 and k t p2 ) and dissociation from the 3'-end (k end p1 and k end p2 ). To help simplify the analysis we assume the same contact size, d, on the ssDNA and that each population translocates with the same translocation kinetic step size, the average number of nucleotides translocated between consecutive rate-limiting steps, m. Given this simplification, we note that the determined contact size and kinetic step size from the analysis represent a lower estimate for both populations. s8 To aid the NLLS analysis we independently measured the Pif1 monomer dissociation rate constant from internal sites of the ssDNA, k d , using poly(dT) and monitoring Pif1 intrinsic tryptophan fluorescence.
Albeit small, the change in the intrinsic fluorescence of Pif1 is sufficient to provide reliable estimate of the signal above noise. A complex of 0.2 µM Pif1 with 30µM nts poly(dT) is mixed in the stopped-flow with different concentrations of ATP and 0.1 mg/mL heparin. Figure S6b shows the resulting time course obtained in Buffer T200 for 0.5mM ATP (final). A single exponential is sufficient to describe the data. The observed dissociation rate constants obtained from the single exponential fit of the data for different ATP concentrations are shown in Figure S6c (red, 200mM NaCl and blue, 100mM NaCl). For ssDNA lattices in which the length is effectively "infinite" given the processivity of the translocase under a given set of conditions the observed rate constant provides an estimate of k d (27). The poly(dT) used in our dissociation experiments has an average length of 300 nts, much larger than the contact site-size of Pif1 (23). Although large, at this stage we cannot assess whether this length is "infinite" for Pif1 translocation, thus our estimate of k d from internal sites should be considered an upper estimate. Indeed, in presence of ATPγS (i.e. no translocation) Pif1 dissociation from the end is faster than from internal sites ( Figure S7).
We constrained the k d for both populations to the determined values ( Figure S6c) for a given ATP and NaCl concentration during the NLLS analysis of the translocation time courses since allowing k d to float for one or both populations resulted in negative estimates for one k d . It is important to note that even if the k d is over estimated (and may not necessarily be the same for both populations) this parameter is not highly correlated with the macroscopic translocation rate constant, m*kt (32,35). Therefore, the reported macroscopic translocation rates are not significantly affected by our k d estimate.
The Cy3 fluorescence time courses for the different lengths of DNA under a given solution condition were analyzed globally using Eq. (e2) and standard NLLS algorithms to obtain estimates of the translocation kinetic parameters (32,35). In the analysis, k t p1 , k t p2 k d , k end p1 , k end p2 , and r were constrained to be global parameters (independent of DNA length); n, P1, and P2 were allowed to float for each time course. In the NLLS analysis the parameter k d was constrained to the value determined independently as discussed above. The kinetic step-size of translocation was then subsequently determined from a linear least squares analysis of the dependence of n on L according to Eq. (e3) ( Figure S8a) (27,32,35).
All NLLS analyses were performed using Conlin kindly provided by Dr. Jeremy Williams (85). The software library CNL50 (Visual Numerics Incorporated, Houston, TX) was used for the numerical calculation of the inverse Laplace transform.

Simulations testing alternative models for Pif1 translocation.
We note that while the model in Scheme 1 captures the second Cy3 fluorescence peak in the time courses it does not describe the first one very well, especially at the higher ATP concentrations and for the higher NaCl concentration. For example, for the longer lengths of ssDNA the initial rise of the Cy3 s9 fluorescence shows a small albeit detectable lag ( Figures S8b). Given the complexity of the translocation model in Scheme 1 expanded for an additional population, alternative translocation models evoking additional steps or altered translocation distributions along the lattice become difficult to assess by our NLLS analysis methods especially when lacking prior information on any of the kinetic parameters.
Therefore, at this stage we provide simulations of different models and discuss their effect on the qualitative behavior of the Cy3 fluorescence time courses. We examined the behavior of the translocation traces collected at 200mM NaCl after mixing with 1mM ATP, where the contribution of the second moving population is minimal. Two different kinetic models were implemented in MatLab and assume Pif1 moves in 1 nt steps with a ssDNA contact size of 6 nts. To simplify the analysis we constrained the second population to follow a n-step sequential model with a random distribution of the protein along the DNA.
Furthermore, we constrained the kinetic parameters to the values determined at 200mM NaCl and 0.5mM ATP (Table S2).
Non-productive to productive conformation transition model-First we considered the possibility that before initiating translocation DNA-bound Pif1 must undergo a transition from a non-productive to a productive complex for translocation. In this case, the presence of one or more steps before translocation would lead to the appearance at the shortest times of a lag or a non-linear dependence of the translocation signal (32,(42)(43)(44)86). We simulated a series of time courses varying the rate constant of the isomerization step and the initial fraction of non-productive translocase for translocation along ssDNA with a random distribution of Pif1 along the ssDNA. Figure S8d  between the non-productive and productive state of DNA-bound Pif1. For these simulations both the fast and slow moving populations of Pif1 are assumed to be initially 100% in the non-productive state and to translocate with a macroscopic translocation rate (m*k t ) of 56 nt/sec (Table S2). When the rate of isomerization is slower than the macroscopic translocation rate, the simulation clearly does not capture the data. For k int of 10 sec -1 the simulation captures the position of the peak but not the initial phase of the time course. Finally, for even larger values of k int the simulations miss both the peak position and the initial part of the time course (note that above 100 sec -1 the simulations become insensitive to the value of the rate constant, i.e. Pif1 is effectively all in the productive state before translocation starts). The situation is not improved when simulations are performed either by varying the macroscopic translocation rate or the fraction of non-productive and productive state of Pif1 ( Figure S8d, middle and right panel respectively). In each case the simulations including the presence of a non-productive to productive transition fail to do a better job in capturing the time course, suggesting that an initial isomerization step is not giving rise to the lag/slow phase in the time course.
Bias Pif1 distribution along the ssDNA lattice-The translocation model in Scheme 1 assumes that Pif1 is initially randomly bound to ssDNA. Therefore we next considered the effect that a small bias of Pif1 either towards the ends or the middle of the ssDNA has on the Cy3 fluorescence time courses (Figure   s10 S9). The fractional distributions of Pif1 along the ssDNA for these simulations are shown in Table S3. For each simulation the translocation parameters are constrained to the ones obtained from analysis of the data with the model in Scheme 1 (Table S2). Only the relative fractions of Pif1 bound to each position on the ssDNA is changed. As reference the fitting according to Scheme 1 with a random distribution of Pif1 on ssDNA is shown as well ( Figure S9b, black trace). From these simulations it is evident that inclusion of either a small fraction of Pif1 bound to the 3'-end (where Cy3 is placed) or a fraction of Pif1 bound to the 5'-end (opposite to Cy3) do not capture the translocation ( Figure S9b, green vs blue). When a fraction of Pif1 is bound to the 3'-end this leads to a higher value of the initial fluorescence and a slight shift of the peak to shorter times. Conversely, when a fraction of Pif1 is bound to the 5'-end the peak is shifted towards longer times as expected when a dominant fraction of Pif1 is bound to the opposite end of the ssDNA. Also, an initial lag would be expected in this case (32,(42)(43)(44)85). However, according to Scheme  Table S1. Substrates with mixed sequence composition used in this study. Figure 3.

Substrates used in
-     Figure S5. a) Time courses of the relative Cy3 fluorescence change after mixing a pre-formed complex of dT 38 -Cy3 and Pif1 (dialyzed in BufferT 100 ) with 1mM ATP and 0.1mg/mL heparin in BufferT 100 (blue) or BufferT300 (red). Also, in black is the mixing of a complex of dT 38 -Cy3 with Pif1 (dialyzed in BufferT 100 ) that is pre-formed in BufferT 200 and then mixed with 1mM ATP and 0.1mg/mL heparin in BufferT 200 . In gray is the same experiment as the black one where however Pif1 was originally dialyzed in BufferT 200 . b) Comparison of Pif1 translocation on either homo-oligodeoxynucleotide dT 28 -Cy3 (red) or the same length of ssDNA composed of a 5'-end dT 8 sequence followed by 20nt of mixed composition (black) (bottom strand in substrates D in Figure 5 and Table S1). c) Pif1 translocation in the presence of 0.5mM ATP (after mixing) on dT 30 T(bio)T 7 -Cy3 (gray) or dT 38 -Cy3 (black) The same experiments performed in the presence of 0.1mM ATP (after mixing) for the same substrates are shown in orange and red, respectively. s22 Figure S8. a) Fitted number of steps, n, in Equation (e2) as function of ssDNA length for the data collected at 100mM NaCl. b) Kinetic step-size as a function of ATP concentration calculated from the linear dependence of the fitted number of steps as function of ssDNA length. b) and c) Fitting according to Scheme 1 (red) of the Pif1 translocation data (black) collected at 200mM NaCl after mixing with 0.1 mg/mL and 1mM ATP (a) and 0.1mM ATP (b). d) Simulations of a model that includes an initial isomerization from a non-productive to a productive state of Pif1. Left panel, both populations in Scheme1 (see Main Text) are initially 100% in the non-productive state and the macroscopic translocation rate, m*k t , is 56 nt/sec. Center panel, both populations are 100% in the non-productive state and the isomerization rate is 1 sec -1 . Right panel, the fraction of non-productive and productive states are indicated in the Figure, the macroscopic translocation rate, m*k t , is 56 nt/sec and the isomerization rate is 1 sec -1 . Figure S9. Simulations of different variants to Scheme 1. The gray dots are the data collect at 200mM NaCl after mixing with 1mM ATP. Color-coding for the simulations is maintained across the different panels. The fitting according to Scheme 1 with a random distribution of Pif1 on ssDNA is shown in black. The data and fits are normalized for clarity. Middle Panel: data for dT 38 -Cy3. Green, simulation for a small fraction of Pif1 having a bias for the 3'-end of the lattice. Blue, simulation for a fraction of Pif1 having a larger bias for the 5'-end of the lattice relative to either a random distribution or the 3'-end. Red, simulation for a fraction of Pif1 having a bias for the middle of the lattice larger than the 3'-end, but smaller than a random distribution. Top Panel: data for dT 28 -Cy3); Bottom Panel: data for (dT 50 -Cy3). Dashed red is a simulation where the fraction of Pif1 bound to the middle of the lattice is allowed to occupy with equal probability the center position and 10 nucleotides to the left or right of it. For more detailed definition of the relative fractions see Table S3.