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. 2010 Aug 20;142(4):544-55.
doi: 10.1016/j.cell.2010.07.016.

PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps

Jeehae Park  1 , Sua Myong, Anita Niedziela-Majka, Kyung Suk Lee, Jin Yu, Timothy M Lohman, Taekjip Ha
Affiliations

PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps

Jeehae Park et al. Cell. .

Abstract

Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 nt. This reeling-in activity requires the open conformation of PcrA and can rapidly dismantle a preformed RecA filament even at low PcrA concentrations, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

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Figures

Figure 1
Figure 1. Repetitive looping of ssDNA coupled with PcrA translocation
A. A dsDNA with 5′-(dT)40 ssDNA tail is immobilized on a PEG surface via the duplex end. B. FRET between the donor (Cy3) and acceptor (Cy5) reports on the change in the time-averaged distance between the tail end and the partial duplex junction. Single-molecule time traces shown (donor intensity in green, acceptor intensity in red, and FRET efficiency in blue) were obtained in the presence of 5 μM ATP and 100 pM PcrA. See also Figures S1A-S1C. C. Cy3 intensity increases as PcrA approaches. This property is used to infer distance change between PcrA and the labeled position. Cy3 intensity time trace was obtained under the same conditions as in (a). D. When Cy3 is attached to the junction no periodic intensity fluctuation was observed upon addition of ATP and PcrA. See also Figures S1D-S1H. E. Reeling-in model (a) PcrA binds at 5′ partial duplex junction. (b) Translocation begins in 3′->5′ direction while PcrA maintain contact at the junction (c) ssDNA loop formation and its increase in size as PcrA continues to translocate toward the 5′ end. (d) PcrA reaches the 5′ end and runs off the track. (b)->(d) is repeated over in continual cycles. F. Representative FRET time traces of PcrA looping 5′pdT40 at varying ATP concentrations. G. Michaelis-Menten fit of repetition rate (1/<Δt>) vs. ATP concentration. Error bars denote STD. Errors in the fit results are shown in SEM.
Figure 2
Figure 2. Repetitive looping is induced by a PcrA monomer
A. Cy3-labeled PcrA and ATP are added to DNA labeled with Cy5 at the end of the 5′ tail and an example is shown for binding at 8 sec, repetitive translocation, and dissociation or photobleaching at 17.7 sec. B. Cy3-labeled PcrA and ATP are added to DNA labeled with Cy5 at the partial duplex junction. An example trace shows only binding and dissociation but no evidence of relative motion between Cy3 and Cy5. C. ATP and Cy3, Cy5 labeled 5′pdT40 DNA are added to PcrA immobilized on the surface via biotinlyated Anti-His-tag-Antibody. An example time trace shows DNA binding at 7.6 sec followed by repetitive looping. See also Figure S2. D. Histogram of time interval of repetition (Δt) shown in C and a gamma distribution fit.
Figure 3
Figure 3. ssDNA length dependence and initiation of looping
A. Representative single-molecule FRET time traces of repetitive looping obtained for 5′ ssDNA tails of various length at 5 μM ATP. See also Figures S3A-S3E. B. Average time of each translocation cycle (<Δt>) vs. 5′ tail length at saturating ATP concentration (1 mM). The number of molecules analyzed is as follows with the average number of repetitions per molecule shown in parenthesis: 20 molecules for 5′pdT40 (110), 14 molecules for 5′pdT50 (104), 30 molecules for 5′pdT60 (73), 36 molecules for 5′pdT70 (54), and 34 molecules for 5′pdT80 (48). The error bar denotes STD. Errors in the fit result are in SEM. See also Figure S3F. C. Before repetitive looping begins, an elevated FRET plateau is observed. D. Histogram of dwell time of the plateau and an exponential fit. The error in the fit result is in SEM.
Figure 4
Figure 4. One nucleotide kinetic step size of translocation
A. A Δt histogram obtained from a single PcrA molecule showing 256 repetitive looping events over 140 sec. A fit to the gamma distribution is shown in (red). Also shown is a fit with the value for N fixed at 10. B. Gamma distribution fits of Δt histograms for 10 different PcrA molecules (R2 > 0.97 for each curve). For clarity, the Δt histograms themselves are not shown. C. N values determined from 10 different molecules shown in B (Error bars denote SEM). The molecules (A to J) are ordered with increasing N values. For the 40 nt translocation, the expected zones for kinetic step sizes of 1, 2, 3 and 4 nt are indicated as shades of different color. D. Heterogeneity among the PcrA molecules. k vs. N for the 10 molecules analyzed in B (Error bars denote SEM). E. Δt histograms and gamma distribution fits obtained from single PcrA molecules on 50, 60, 70 and 80 nt 5′ tail of partial duplex DNA. F. N vs. ssDNA length for the molecules shown in A and E (Error bars denote SEM).. G. k vs. ssDNA length for the molecules shown in A and E (Error bars denote SEM).. H. Δt histogram obtained from 77 molecules and gamma distribution fit (R2=0.99).
Figure 5
Figure 5. PcrA on a forked DNA and the role of 2B domain of PcrA
A. A forked DNA substrate labeled with donor and acceptor at the same position as in Fig. 1A. A representative set of traces of fluorescence intensities and FRET efficiency are presented before and after adding PcrA (100 pM) and ATP (5 μM). B. Number of molecules per imaging area before and after adding PcrA (100 pM) and ATP (1mM) (Error bars denote STD). C. Representative time traces of 5′pdT40 in the presence of G423T mutant PcrA and 1 mM ATP. See also Figures S4A-S4C. D. FRET values at every time point of the trajectory are collected and plotted into a histogram (wild type in red, G423T in green). E. Dual-labeled PcrA is added to unlabeled 5′pdT40 with 1 mM ATP. F. Representative time trace of fluorescence intensities and FRET for dual-labeled PcrA. Every one second, a 0.1 second pulse of red illumination (633 nm) and no green illumination (532 nm) is applied in order to verify the presence of acceptor. Periods of green illumination are marked with green bars and red illumination with red bars above the time traces as well as with shades over the traces. See also Figures S4D-S4G.
Figure 6
Figure 6. RecA filament removal by PcrA
A. and B. 5′pdT40 DNA is pre-incubated with 250 μM RecA and 1 mM ATP for 5 min. FRET histograms are shown before and after incubation. See also Figures S5A-S5C. C. and D. 1–40 nM PcrA is then added to the pre-formed RecA filament maintaining ATP and RecA concentration constant in solution. FRET histograms were obtained 10 min after PcrA addition. See also Figure S5D. E. Fraction of RecA remaining vs. time for different PcrA concentration conditions. See also Figure S5E-S5F. F. Fraction of DNA molecules that lost RecA filament 10 min after PcrA addition (Error bars denote SEM from the exponential fit of Fig. 6E). G. Representative time traces of fluorescence intensities and FRET efficiency upon adding 1 nM PcrA addition to the preformed RecA filament. See also Figures S5G-S5H.
Figure 7
Figure 7. Structural model of PcrA translocation on 5′ ssDNA of partial duplex
A. Crystal structure of PcrA in closed form in complex with a 3′-tailed partial duplex DNA. PcrA is in the Van der Waals representation with the domains colored individually in yellow (1A), green (2A), purple (1B) and orange (2B). DNA is presented with two colors for each strand: brown (3′ tail strand) and pink (5′ complementary strand). B. Open form of PcrA generated by 130° rotation of 2B domain together with DNA from the structure shown in A. C. The ssDNA binding sites of PcrA in the closed form as shown in A. Each protein residue is colored according to the color scheme of the domain it belongs to. D. The ssDNA binding sites of PcrA in the open form as shown in B. Color schemes are the same as those used in C. Inset shows an enlarged image of the region around 5′ end of the DNA placed in proximity to F192. E. PcrA translocation model at the DNA fork. The closed form of PcrA is shown inside the pink circle. Others are in the open form. Movement of PcrA is indicated with green arrows. The relative movement of the 5′ ssDNA tail (pink) is shown with pink arrow. See also Figure S6.
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