A mechanistic study of helicases with magnetic traps

Abstract

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue.

Keywords: DNA unwinding; Holliday junction migration; active unwinding; ds-DNA fork; fork regression; helicase/polymerase coupling; helicases; magnetic traps; polymerase; primase; primosome; replisome; strand annealing.

Figure 1

Possible translocation mechanisms of helicases. (A) The helicase can be passive, trapping fork fluctuations through an ATP‐driven progression. Alternatively, helicases can actively unwind DNA (B) as they progress along by a “rolling model” where each monomer takes turn at unwinding the molecule or (C) by driving a “plow” through the DNA fork or (D) pulling the DNA through the helicase as in some hexameric helicases. So far all these mechanisms are found in helicase with the exception of the rolling model which had not been demonstrated experimentally for any helicase.

Figure 2

(A) The flow cell introduced by the Kowalczykowski group to study the interaction between DNA and various motors. It consists of a microfluidic cell with two (or more) inlets. Through one inlet a solution of ATP, dye, Mg⁺⁺, etc. can be introduced and through the other, DNA bound to transparent beads and possibly proteins (such as RecBCD). In this laminar flow regime, the two solutions do not mix. One bead is captured by optical tweezers. The cell is then translated and the trapped DNA molecule brought in contact with ATP (to launch the unwinding reaction) and/or a dye (to stain the molecule). (B) as the molecule is unwound by the progression of RecBCD, it is degraded by the RecB exonuclease activity. Its length can be deduced by the fluorescence of the stained and stretched remaining dsDNA. Hence the progression of the helicase complex on the molecule can be monitored in real time. (Figure taken from Figure 1 of [32] with permission)

Figure 3

(A) Schematics of the unpeeling configuration used to study helicases: a helicase (blue blob) loads on a nick or gap in the dsDNA molecule under tension. Unwinding of the molecule results in an increase (Δz = z′ – z) of the overall extension. The two ssDNA (one under tension and one free) are unable to match in the wake of the enzyme due to a mismatch in their extension. (B) Schematics of the unzipping assay: a helicase (the violet hexamer) loads at the fork of a DNA hairpin under tension. Unwinding of the hairpin results in an increase (Δz = z′ – z) of the molecule's extension. The tension on the released ssDNA strands prevents their reannealing in the wake of the enzyme. As the helicase reaches the hairpin apex, its continuing translocation on a ssDNA template allows for reannealing of hairpin in its wake, monitored by the decreasing change in extension (Δz = z″ – z)

Figure 4

Extension of a DNA hairpin molecule versus the applied force in the unzipping configuration. The molecular design is given in the box on the right: a 1.2 kb hairpin is made of a dsDNA stem closed at one end by a loop and with a fork at the second end. The arm ending in 5′ has a biotin while the other arm holds multiple digoxigenin. This molecule is attached to a 1‐micron size magnetic bead coated with streptavidin and to its second extremity to the flow cell via a digoxigenin/anti‐dig bound. At low force this molecule remains closed and its extension is null. As the magnets are brought closer to the bead, the force can exceed 15 pN leading to the molecule unfolding. The DNA sequence of this molecule presents a GC rich zone close to the apex: this region is more difficult to open and remains folded until the force reaches 17 pN. At that force the molecule is fully open and its extension is nearly 1.2 μm. Further increase of the force leads to a small stretching of the ssDNA molecule. Upon decreasing the force, the molecule refolds with a hysteresis of typically 3 pN. The refolding process nucleates at the molecule apex. If one introduces a 18 nts oligonucleotide that hybridizes to the apex, the refolding of the hairpin is hindered and one observes the red curve corresponding to ssDNA elasticity. When the force is decreased to very low values, the oligonucleotide can be expelled and the hairpin refolds

Figure 5

Right, schematic representation of a magnetic trap. Small super‐paramagnetic beads are bound to the surface of a flow cell by one (or a few) DNA molecules. Magnets positioned above the sample exert a force on the beads and thus on the tethering molecules. DNA hairpins attached at their free ends by one strand to a magnetic bead and at the opposite one to a surface can be unzipped at high enough force (typically F > 15pN). About 50 beads are simultaneously tracked on an inverted microscope. A typical image is shown on the left. Analysis of the successive images of the beads on a camera allows deducing their 3D position and from it the distance of the bead to the surface (i.e., the extension of the tethering molecule) and the exerted force (from the bead's fluctuations, see text)

Figure 6

A typical signal observed with UvrD in the unzipping configuration: Unwinding (U) of the dsDNA results in a continuous increase of the extension by N _U bps during a time τ _U from which the mean processivity < N _U>, mean rate of unwinding v _U =<N _U/τ _U> can be determined. Unbinding of the enzyme results in re‐hybridization (H) of the two strands. Strand switching by UvrD results in an ATP dependent reZipping (Z) of the two strands in the wake of the helicase

Figure 7

(A) More complex signals observed with UvrD resulting from a combination of the elementary processes described in the text: Unwinding (U), re‐zipping (Z). Signal obtained in the unzipping configuration

Figure 8

(A) Signal produced by a UvrD helicase unwinding a 1.2 kbps DNA hairpin at T = 29°C and F = 11.5 pN. Before t = 4078 s the hairpin is closed. Then (1) a UvrD helicase loads on the hairpin and starts unwinding it in a processive and fast rate (about 380 bp/s). The UvrD helicase reaches the hairpin apex (2) and pursues its translocation as it essentially travels on a ssDNA molecule. Once the helicase has passed the apex, the hairpin starts refolding until the fork bumps on the helicase (3). The extension in this phase reproduces the position of the helicase as it translocates along ssDNA at a rate of about 400 bp/s. Note that the motion is extremely regular (each point corresponds to a video frame acquired at 30 Hz). (B) A trace obtained in conditions similar to (A) but where the helicase changes speed as it re‐zips the molecule. The events with lower speed are not very frequent but do occur repeatedly, suggesting that a helicase might display different rates perhaps due to a change in its conformation. (C) Unzipping and re‐zipping rates show no significant dependence on the applied force (the gray area corresponds to forces where the statistics on traces is weaker)

Figure 9

(A) Comparison of the rates of unwinding of RecQ and gp41 as a function of the tension on the hairpin molecule in the unzipping assay at saturating ATP concentrations. (B) Comparison of the rates of unwinding of RecQ and gp41 as a function of the AT content of the hairpin molecule (at F = 9 pN). The independence of the rates of unwinding by RecQ on force and AT content are compatible with an active unwinding mechanism. On the other hand, the strong dependence of the rates of unwinding by gp41 on force and AT content are indicative of a passive unwinding mechanism, whereby the helicase progression is limited by the probability of spontaneous fork opening.52

Figure 10

To investigate the translocation of a helicase on ssDNA the following assay has proved useful. Once the enzyme has started to unwind the hairpin fork, the force is increased such as to mechanically unzip the hairpin. The enzyme thus proceeds by translocating on ssDNA for a time T _SS determined by the time span over which the hairpin is maintained open. Upon reducing the tension of the DNA molecule, the hairpin rewinds until its fork encounters the helicase. At that point the increase in the DNA's extension, N bpS, allows one to deduce the rate of translocation of the enzyme on ssDNA: v _SS= N bp/T _SS

Figure 11

Models of primosome behavior during primer synthesis. (A) In the T4 virus, the helicase and the primase work as a complex encircling the lagging strand. However, their respective displacement directions are just opposite raising the question of how they collaborate. (B, C, and D) Schematic representation of three possible models for helicase and primase interaction during primer synthesis (left) and the real‐time DNA extension traces expected for each model (right). (B) In the pausing model the helicase temporarily stop translocating during priming. (C) In the disassembly model the primase dissociates from the helicase to synthesize a primer while the helicase continues unwinding DNA. (D) In the DNA looping model the primosome remains intact and DNA unwound during priming forms a loop

Figure 12

Primer synthesis by primosome depends on rNTP concentration. (A) Experimental traces corresponding to the gp41 helicase activity (green) and the wt primosome activity (red) in the absence of rNTPs. (B) Experimental trace displaying characteristics (unwinding velocity during priming, position and lifetime of the block) for the primosome disassembly model. DNA looping mechanism for primer synthesis. (C) Experimental trace displaying characteristics (unwinding velocity during priming and the loop size) for the DNA looping model.68

Figure 13

Single molecule studies of the coupling between the T4 helicase (gp41) and the T4 polymerase (gp43). (A) At low forces (F < 7pN), the polymerase synthesizes the complementary strand as the helicase unwinds the molecule, resulting in a fast increase (red) of the molecule extension. As the helicase and polymerase meet head‐on at the hairpin apex, they stall (black). The helicase then falls off and the polymerase resumes its activity on the remaining ssDNA (the upper complementary strand of the hairpin). (B) At higher forces (F > 7pN), the helicase unwinds DNA (blue) independently of the proximity of the polymerase which synthesizes the complementary strand on the unwound ssDNA. As the helicase falls off (at t = 30 s), the hairpin re‐hybridizes up to the point where the polymerase is (the dashed line represents the polymerase rate during the uncoupled helicase unwinding). The polymerase resumes polymerization in the very inefficient strand displacement mode (green). (C) Measured rates of unwinding and strand‐synthesis by gp41 and gp43 alone or when they are coupled (with or without the SSB protein, gp32). Notice that at low forces (F < 5pN) and in the absence of helicase the polymerase switches into exonuclease mode (its rate is negative).76

Figure 14

(A) The substrate for the single molecule study of the rewinding activity of a helicase is an opened hairpin with a small stem loop at its end (that serves as a loading site for the enzyme). (B) Upon enzymes rewinding the extension of the hairpin (maintained under a high tension) is observed to decrease. Sudden increases in extension are due to dissociation of the enzyme from its substrate and subsequent force‐induced unfolding of the hairpin.80

Figure 15

Reconstruction of the template switching pathway. (A) Construction of a stalled fork substrate with a LNA block at the leading strand, mimicking a damage. (B) Experimental trace at F = 8 pN displaying the extension z(t) with ATP, dNTPs, UvsW and T4 holoenzyme starting with the stalled fork and ending with the fully replicated substrate. (C) Schematic of the repair of a stalled replication fork by its regression.79

Figure 16

Upf1 helicase activity on RNA. (A) Organization of Upf1 and truncated versions (gray arrows) used in our study, Upf1‐HD and Upf1‐CH‐HD. (B) Schematic representation of the RNA used for the MT set up. (C) Experimental trace showing the activity of Upf1‐HD in saturating concentration of ATP. The number of unwound bases is deduced from the molecular extension Z(t) obtained at F = 10pN. From 5600 s to 6440 s, the helicase unwound the 156bp RNA hairpin. From 6640 s to 7200 s, the RNA hairpin refolded, while Upf1‐HD translocated on ssRNA reaching the 3′extremity. (D, E, and F) The binding of Upf2 to CH domain activates Upf1‐CH‐HD unwinding and translocation. (D, E) Experimental traces corresponding to the two types of enzymatic activity detected for Upf1 on a DNA hairpin. (D) Upf1 binds on ssDNA (starting at 231 s), blocking the re‐zipping of the hairpin. (E) The enzyme is active. (F) Histogram of relative activity of Upf1‐CH‐HD, Upf1‐CH‐HD/Upf2 complex and Upf1F192E mutant. (G) Trace of human Upf1‐CH‐HD/Upf2 complex unwinding steadily and completely the DNA molecule, passing the apex, pursuing its translocation and refolding the DNA hairpin. At t = 675 s, the complex makes two strand switching events before finally stopping its activity leaving the hairpin blocked (at t = 687 s). This trace is atypical since the Upf1‐CH‐HD/Upf2 complex translocates for some time before stalling; for most bursts, the complex stops very quickly after passing the apex. (H) Distribution of instantaneous unwinding rate of the Upf1‐CH‐HD/Upf2 complex.87