Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase

Abstract

An encounter between a DNA-translocating enzyme and a DNA-bound protein must occur frequently in the cell, but little is known about its outcome. Here we developed a multicolor single-molecule fluorescence approach to simultaneously monitor single-stranded DNA (ssDNA) translocation by a helicase and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA-binding properties, the two cognate ssDNA-binding proteins RPA1 and RPA2 differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.

Figure 1. Calibration of Cy3 fluorescence quenching by the FeS cluster of XPD helicase

(A) Experimental design: The quenching experiments were carried out under stoichiometric binding conditions. When XPD and the ssDNA substrate were present in equimolar concentrations, XPD binding positions were uniquely dispersed along the 42mer oligonucleotide labeled with Cy3 at one end. The ensemble-average position was expected to coincide with the center of the oligonucleotide. When XPD was present at two-fold excess concentration over the DNA substrate, we expected two helicase molecules to be bound to each oligonucleotide occluding its complete length. Thus, for each helicase: ssDNA ratio, we predicted the distance between the front edge of the helicase and Cy3. (B) Quantification of the quenching magnitudes (blue circles). The solid line represents linear approximation of Cy3 quenching as a function of the distance between the leading edge of XPD and the dye.

Figure 2. Cy3 quenching allows visualization and analysis of XPD binding to and translocation along ssDNA

(A) Schematic depiction of the immobilized ssDNA (42mer) with Cy3 at the 5’-end and a representative single-molecule trajectory of XPD-dependent Cy3-quenching in the presence of ATP (right panel). Cy3 fluorescence intensity abruptly decreased as the result of XPD binding and then gradually recovered as the helicase translocated away from the dye in the 5’→3’ direction. (B) Surface-tethered ssDNA (42mer) labeled with Cy3 at the 3’-end. A representative fluorescence trajectory shows that XPD translocation along this substrate resulted in gradual quenching of Cy3 intensity followed by its full recovery when XPD dissociated from the substrate. (C) Representative fluorescence trajectories for the substrates of 22, 32 and 42 nucleotides long carrying the Cy3 dye at the 3’-end depict the length-dependence of the binding/translocation patterns. (D) Effect of XPD concentration on the frequency of the observed events (red) and on the rate of XPD translocation (blue). (E) Both frequency of observed translocation events (red) and translocation rate (blue) were functions of ATP concentration.

Figure 3. The two F. acidarmanus RPAs differentially affect XPD binding to and translocation along ssDNA

(A) In the case of XPD translocation on protein-free ssDNA, 138 individual translocation events were observed in 600 analyzed trajectories. Individual rates were binned in 5% fluorescence change per second intervals, plotted and analyzed by fitting the resulting histogram to normal distribution. The average and standard deviation were used to calculate the average translocation rate. (B) Only 48 events were observed and analyzed per 600 trajectories recorded in the presence of 100 nM RPA1. (C) In the presence of 100 nM RPA2, the frequency of observed events slightly increased (193 events were observed and analyzed per 600 traces). The histogram displayed two peaks and was fitted with double normal distribution resulting in the two characteristic rates. Representative fluorescence trajectories for each condition are shown on the right of respective histograms. (D) Fractions of XPD molecules translocating at slow rates were calculated as the ratio of area under the left hump of double Gaussian distribution to the total area under the distribution curve. (E) Kinetic association constant, k_on was calculated from the frequency of the quenching events recorded at 150 pM XPD. Error bars represent standard deviation for three sets of 100 randomly selected trajectories.

Figure 4. Two observed modes of XPD translocation along RPA coated ssDNA

(A) Binding of the Cy5-labeled RPAs to the Cy3-labeled ssDNA (schematically depicted on the left) can be detected by following FRET between the two dyes. Fluorescence of the Cy3 and Cy5 dyes is shown in green and red, respectively. (B) If RPA (100 nM) is displaced by the translocating XPD helicase (150 pM) or dissociates from ssDNA, we expected to observe uncoupled fluorescence trajectories. Examples of the fluorescence trajectories displaying such a behavior are shown for ssDNA coated with both RPA1 (middle) and RPA2 (right). (C) If XPD (150 pM) translocates over the bound RPA (100 nM) without displacing it from ssDNA, we expected to observe a synergistic quenching followed by the recovery of Cy3 and Cy5 fluorescence.

Figure 5. Translocation of XPD observed through quenching of directly excited Cy5- RPA1 and 2

(A) Visualization of ssDNA binding by 5 nM Cy5-RPA. The ssDNA binding/dissociation of either RPA1 (middle) or RPA2 (right) was monitored by following change in Cy5 fluorescence intensity (the “on” and “off” states are indicated by respective dotted lines). (B) The effect of XPD translocation on DNA bound RPAs. Two distinct types of Cy5 fluorescence trajectories were observed in the presence of 150 pM XPD and 1 mM ATP. In the type 1, the Cy5 fluorescence was quenched and recovered resulting in a symmetric trajectory (Type 1). Gradual decrease and increase of Cy5 intensity reflected XPD approaching (arrow 1) and moving away (arrow 2) from Cy5 labeled RPA. In the second type of trajectories we observed only gradual quenching of Cy5 and no recovery (Type 2).

Figure 6. XPD facilitates displacement of RPA1 but not RPA2

(A) Representative FRET trajectory of Cy5-RPA1 binding and dissociating from Cy3-labeled ssDNA. The “on” and “off” states for the Cy5 signal (red) are indicated on the right. The duration of the ssDNA bound state of Cy5-RPA1 is indicated by the arrow. (B) Representative FRET trajectory of Cy5-RPA1 recorded in the presence of 150 pM XPD and 1 mM ATP. The duration of the ssDNA-bound state of Cy5-RPA1 just before XPD translocation was measured as indicated by the arrow. (C) Average duration of the XPD “on” state. The error bars represent standard deviation for 25 individual events.

Figure 7. Model for XPD targeting to RPA2 coated ssDNA and its translocation

XPD is schematically represented by the light blue figure. OB-folds (RPA1 and RPA2) and Zn-finger (RPA1) domains are depicted as green crescents and yellow circles, respectively. Stretching of the ssDNA molecule by RPA1 may inhibit XPD–ssDNA interaction, while ssDNA that is pre-bent due to the interaction with RPA2 represents a substrate that can be accommodated between the two ssDNA binding sites on XPD. Subsequent progression of XPD along a ssDNA lattice has to occur either by displacing bound RPA or translocation over it. The small binding site size of RPA2 and its dynamic association with ssDNA may result in ssDNA continuously occupied by ssDNA-binding proteins during XPD translocation.