Two steps forward, one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

Abstract

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5'-3' polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein-DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).

Keywords: DNA helicases; DNA repair; FeS cluster; High-resolution optical tweezers; Nucleotide excision repair; Single-molecule; Total internal reflection fluorescence microscopy (TIRFM).

Figure 1. Modular organization of FeS DNA helicases

A. All XPD-like helicases contain the motor domains (HD1 and HD2) and auxiliary domains (FeS and ARCH). Helicase signature motifs are indicated by black bars and Roman numerals. Two additional domains found in FANCJ, CHLR1 and RTEL are shown in green. Most studied mutations in the auxiliary domains of XPD-like enzymes are shown in red (breast cancer causing mutations in FANCJ), blue (Fanconi anemia-associated mutation in FANCJ), purple (TTD-associated mutations in XPD) and green (mutations that disable the damage verification site). B. Structure of XPD (PDB: 4a15) is shown as ribbon diagram. Individual domains are colored as following: HD1 is salmon, HD2 is blue, FeS domain is brown and ARCH domain is purple. Five nucleotide ssDNA bound to the non-canonical site on HD2 resolved in the PDB: 4a15 structure are shown in red space filling model. From the structural superimposition of the motor cores XPD with another SF2 helicase (PDB: 2db3, Vasa DEAD-box RNA helicase bound to ssRNA) the ssDNA can be extended into the canonical DNA binding grove by additional 5 nt (green space filling model). AMP-PNP, an ATP analog is also from the PDB: 2db3 (yellow space filling model). C. Cartoon depiction of the XPD bound to DNA and ATP. Green and purple spheres correspond to mutations highlighted in (A); orange spheres correspond to the key residues within the extended DNA binding site that control helicase activity. References in the callouts indicated the manuscripts that laid the basis for each highlighted element of the model (see text for the details).

Figure 2. FeS cluster is a built-in proximity indictor

A&B. Schematic depiction of utilization of the FeS cluster in XPD to monitor ssDNA translocation and unwinding, respectively. C. Distance-dependence of the fluorescence quenching magnitude. D. One-color TIRFM experimental setup for monitoring XPD translocation. Excitation by TIR and resulting evanescent wave (green arrow) illuminates a thin layer of the flow cell and excites Cy3-labeled DNA molecules tethered to its surface. Fragment of the movie frames is shown as the insert. In it, each green spot corresponds to a single surface-tethered DNA molecule. E&F. Fragments of the representative fluorescence trajectories for the helicase moving away and towards the label, respectively. The data shown were adapted from [54].

Figure 3. Analysis of XPD translocation trajectories

A. Four distinct classes of unwinding events routinely observed in XPD translocation experiments. The top three classes could be fitted with multi-segment lines to determine the translocation rate. The fourth trajectory depicts two translocation events that occurred too close to one another to be analyzed. B. The rates of individual translocation events are binned and plotted as a histogram whose Gaussian fit yields average translocation rate. C. XPD concentration dependence of the frequency of the observed events and XPD translocation rates suggests that at the XPD concentrations below 200 nM we were observing translocation by individual XPD monomers. D. Both the translocation rate and the frequency of the observed events display Michaelis-Menten dependence on ATP concentration. The data shown were adapted from [54].

Figure 4. Analysis of XPD stepping kinetics in the high-resolution optical tweezers experiment

A. The construct for monitoring XPD helicase activity consisted of a dumbbell DNA structure, in which two 1.5 kb handles were tethered to the polystyrene beads held in the optical traps, and an 89 bp hairpin structure flacked by a ssDNA region that served as a loading site for XPD. Blue arrow depicts direction of the XPD movement along the hairpin. Each unwound base pair lengthens the construct by 2 nucleotides (shown in red). The substrate is constructed in a way that allows only the hairpin to be unwound and not the handles (see [67] for details). B. A representative unwinding trajectory. After dumbbell incubation in the XPD containing channel (1) the construct is transferred into the ATP-containing channel (2) where unwinding is detected under the constant applied force as the change in the length of the dumbbell structure resulting in the change in the position of the right bead. This particular trajectory shows 5 consecutive attempts at hairpin unwinding by a single XPD molecule. Each unwinding burst is followed by sliding of the helicase back to the hairpin base. C. A fragment of the representative unwinding trajectory with actual extension data (grey) overlaid with filtered data (blue) and steps determined from fitting the data (red). D. Representative scatter plot of step pairs. Each data points represent the size of every adjacent pair of steps. Consecutive forward steps at highlighted in red, the pairs consisting of at least one backward step are in orange, and 5-bp steps are in green. The data shown were adapted from [67]. E. Proposed mechanics of 5-bp stepping.

Figure 5. Obstacle bypass by XPD helicase visualized in pseudo-tricolor TIRFM experiment

A & B. Schematic representation of the experimental setups for simultaneous detection of XPD and Cy5-labeled RPA2. In (A), the presence of RPA2 on ssDNA is detected due to FRET between Cy3 on DNA and Cy5 on RPA2, while XPD binding and translocation due to FeS mediated fluorescence quenching of both Cy3 and Cy5. In (B), Cy5-RPA2 fluorescence is excited directly. Note that despite of Cy5-RPA2 abundance in the solution, only molecule bound to the surface-tethered DNA produce fluorescence signal (inset). C. Four schemes for detecting XPD translocation (i) on protein-free DNA, (ii) on the protein-coated ssDNA but with only XPD detection, (iii) on the protein coated ssDNA with simultaneous detection of both ssDNA and RPA2, and (iv) by directly exciting RPA2. Representative fluorescence trajectories and rate distributions are shown on the right. The data shown were adapted from [54].

Figure 6. Building an active helicase

Two models for cooperation between XPD and RPA2 A. Scenario 1: RPA2 (depicted in green) binds at the ss-dsDNA junction, destabilizes 4–5 bp dsDNA upstream of the helicase. XPD then advances into the melted region trapping the released strands. Bound RPA2 moves with the translocating strand through the motor core of XPD. The cycle repeats with the next RPA2 molecule (yellow 2&3) binding at the junction. The prerequisite for this scenario is opening of the ARCH domain sufficient to accommodate RPA2-ssDNA complex. B. Alternatively, The same molecule of RPA2 may remain associated with the helicase jammed between the junction and the hole through which the translocating strand passes. Additional RPA2 molecules (2&3) may trap the unwound strands behind the helicase.