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Direct observation of structure-function relationship in a nucleic acid–processing enzyme

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Science  17 Apr 2015:
Vol. 348, Issue 6232, pp. 352-354
DOI: 10.1126/science.aaa0130

Engineering superenzyme function

Understanding how protein domains and subunits operate is critical for engineering novel functions into proteins. Arslan et al. introduced intramolecular crosslinks between two domains of the Escherichia coli helicase Rep, which unwinds DNA. By inserting linkers of different lengths, the domains can be held either “open” or “closed.” The closed conformation activates the helicase, but it can also generate super-helicases capable of unzipping long stretches of DNA at high speed and with considerable force. Comstock et al. used optical tweezers and fluorescence microscopy to simultaneously measure the structure and function of the bacterial helicase UvrD. They monitored its DNA winding and unwinding activity and its shape during these activities. The motor domain also has a “closed” conformation during DNA unwinding and switches to a reversed “open” conformation during the zipping-up interaction.

Science, this issue p. 344 and p. 352

Abstract

The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed “closed” and “open,” correlate with movement toward or away from the DNA fork.

Helicases are vectorial enzymes that play a critical role in genome maintenance, hydrolyzing nucleotide triphosphates to translocate along nucleic acids and separate the duplex strands. UvrD (DNA helicase II) is a prototypical superfamily 1 (SF 1) helicase involved primarily in nucleotide excision repair and methyl-directed mismatch repair in Escherichia coli (1, 2). Although a UvrD monomer is known to translocate along single-stranded DNA (ssDNA) in a 3′-to-5′ direction (36), studies indicate that highly processive duplex DNA unwinding requires at least a dimer (4, 711). UvrD and structurally related SF 1 homologs (including Rep and PcrA) consist of four domains (1216): the RecA-like motor core domains 1A and 2A and the accessory domains 1B and 2B (Fig. 1). Previous studies have shown that the 2B domain of UvrD (14, 16) and homologs (12, 13) can exhibit two orientations—“open” and “closed” relative to the other domains (Fig. 1)—believed to regulate activity (6, 9, 14, 16). Despite detailed structural and biochemical data (4, 911, 14, 17), a lack of direct evidence linking the UvrD conformational state to its function has prevented a more complete understanding of its mechanism. In this study, we used an instrument combining high-resolution optical traps and single-molecule confocal microscopy to measure simultaneously the unwinding activity and conformation of the UvrD helicase (fig. S1) (18, 19). Through these direct measurements, we demonstrate the link between UvrD oligomeric state and processivity, as well as between conformational state and unwinding versus rezipping activity.

Fig. 1 Experimental layout.

Structure of the UvrD monomer in the open (upper right) [Protein Data Bank identification number (PDB ID) 3LFU] (16) and closed (middle) (PDB ID 2IS2) (14) conformations with single-fluorophore location. Two microspheres (gray) in dual optical traps (orange cones) are tethered together by a DNA hairpin. One or more UvrD monomers bind ssDNA and unwind the hairpin in the presence of ATP. A confocal microscope (green cone) detects the configuration of the same fluorescently labeled UvrD unwinding complex.

We monitored helicase activity by detecting the unwinding of a DNA hairpin with an optical trap, as described previously (20, 21). The trap maintained a constant tension (4 to 15 pN; 10 pN for data shown unless otherwise specified) (19) and tracked the number of hairpin base pairs unwound. The composition and conformation of the same UvrD unwinding complexes, site-specifically labeled with one or two fluorophores (with labeling efficiencies ranging from 71 to 85%) (19), were detected with the confocal microscope (Fig. 1).

We observed two distinct types of DNA duplex unwinding activity, which we termed “frustrated” and “long-distance.” During frustrated activity (Figs. 2A and 3, B and C, lower panels), UvrD exhibited repetitive, bidirectional motion on DNA, during which <20 of the available hairpin base pairs unwound and rezipped [13.6 ± 1.8 base pairs (bp), mean ± SEM unless otherwise noted]. This bidirectional activity is distinct from the repetitive ssDNA translocation observed previously with UvrD (6) and its SF 1 homologs (22, 23). Reversals in direction occurred frequently (mean unwinding and rezipping durations 0.25 ± 0.01 s and 0.23 ± 0.02 s, respectively), both midhairpin and after complete hairpin rezipping, and typically repeated many times before UvrD dissociation (9.2 ± 1.2 repetitions for 7.8 ± 1.3 s). In contrast, during long-distance activity (Fig. 2B, lower panel), UvrD systematically unwound >20 bp (38.9 ± 5.6 bp, on average). UvrD motion was far less repetitive, although reversals in direction did occur (mean unwinding and rezipping durations 2.82 ± 0.30 s and 1.38 ± 0.13 s, respectively) at midhairpin [e.g., at 33 s (Fig. 2B)], at the end of the hairpin upon complete unwinding [89 bp (fig. S2)], and after full hairpin rezipping. The mean rezipping speed was dependent on adenosine triphosphate (ATP) and nearly the same as for unwinding (fig. S3), strongly suggesting active translocation of UvrD. An alternate in which rezipping results from backsliding (14, 21) would predict nearly instantaneous rezipping events, inconsistent with the data. Because UvrD is a strict 3′-to-5′ ssDNA translocase (4) with tight ATP coupling (24), rezipping must correspond to 3′-to-5′ translocation away from the ssDNA-dsDNA junction (dsDNA, double-stranded DNA), allowing the duplex to base pair in its wake. Thus, reversals in direction are probably the result of switching ssDNA strands, as first proposed by Dessinges et al. (25).

Fig. 2 Effect of UvrD oligomeric state on DNA unwinding activity.

Representative time traces of unwinding activity for a UvrD monomer (A) and dimer (B), respectively. (Upper panels) Fluorescence photobleaching from a monomer (A) and a dimer (B) (240 and 120 ms per point, respectively). (Lower panels) Simultaneous measurements of hairpin unwinding (U) and rezipping (Z). The UvrD monomer displays frustrated unwinding (A) (15 bp unwound), whereas the dimer displays long-distance unwinding (72 bp) (B). (C) Histogram of the maximum number of base pairs unwound per unwinding attempt (N = 401 replicates), showing frustrated (<20 bp) and long-distance (>20 bp) unwinding. (Inset) Distribution of fluorophore count for frustrated (blue) or long-distance (red) unwinding attempts.

Fig. 3 Effect of open versus closed UvrD conformation on unwinding activity.

(A) Location of donor and acceptor fluorophores for FRET measurement and model of UvrD conformational switching. Upper (and lower) orange arrows denote 2B (and 1A-2A) domain orientation. See text and (19) for details on the model. (B and C) Representative time traces of monomeric UvrD conformation and activity. (Upper panels) Donor (green) and acceptor (red) fluorescence intensity. (Middle panels) Corresponding FRET efficiency showing UvrD reversibly switching between open (low FRET) and closed (high FRET) conformations (dashed red lines). Fluorescence and FRET data are integrated to 60 and 50 ms per point for (B) and (C), respectively. Shaded and unshaded areas denote low- and high-FRET intervals, respectively. (Lower panels) Simultaneous measurements of unwinding (U) and rezipping (Z) of the DNA hairpin. Fluorescence intensity and frustrated unwinding activity are consistent with monomeric UvrD-DNA complexes (which dissociate at 6.2 and 1.8 s). (D) Correlation between UvrD activity and conformation. The mean FRET efficiency and mean UvrD velocity determined over each time interval are plotted (white points; N = 109 intervals, 15 molecules). The color map represents the probability distribution of FRET state and velocity (19).

Our claim that frustrated and long-distance unwinding are distinct activities of UvrD is supported by measurements of the number of consecutive base pairs unwound per unwinding attempt (Fig. 2C) (19), which show a dramatic decrease past ~20 bp. Moreover, the two distinct activities correlate with the number of UvrD helicases present on the DNA hairpin, measured by counting singly labeled UvrD (19). When we observed frustrated unwinding activity, we usually detected only a single fluorophore, indicating a single UvrD loaded (Fig. 2A, top panel). In contrast, when we observed long-distance unwinding, we were more likely to detect two fluorophores, indicating two UvrD helicases (Fig. 2B, top panel). These trends were corroborated over many UvrD-DNA complexes (Fig. 2C, inset). These observations are consistent with previous reports that UvrD dimers are required for long-distance unwinding (4, 79), but they also demonstrate that monomers are competent to unwind a limited amount of DNA under tension (fig. S4) (19).

The ability of a single UvrD helicase to unwind and rezip DNA reversibly many times and in succession suggests that it can switch strands without dissociating from its DNA substrate. Previous studies showed that beyond the 1A-2A motor domains contacting ssDNA, the 2B domain can contact the junction duplex (6, 14). We thus used our instrument to detect the unwinding activity of individual UvrD monomers via optical trapping simultaneously with conformational changes of the 2B domain via single-molecule fluorescence resonance energy transfer (FRET) (26). UvrD was labeled with donor and acceptor fluorophores such that high or low FRET efficiency revealed the closed or open conformation, respectively (16, 19) (Fig. 3A).

Two example data traces of UvrD monomer conformation and unwinding activity measured simultaneously are shown in Fig. 3, B and C. The bottom panels show DNA duplex unwinding and rezipping, as detected by the optical trap. The top panels show highly dynamic donor (green) and acceptor (red) fluorescence signals during activity. In the middle panels, the corresponding FRET efficiency fluctuates between high and low values (0.7 ± 0.2 and 0.3 ± 0.01, respectively, for all molecules), consistent with the closed and open conformations, respectively (16). Dividing the unwinding and fluorescence data traces into time intervals determined by FRET state (i.e., conformation) reveals a correlation between unwinding versus rezipping activity and 2B orientation. When a monomer is in the closed conformation (Fig. 3, B and C, shaded intervals), the DNA duplex unwinds, whereas the duplex rezips upon switching to the open conformation (Fig. 3, B and C, unshaded intervals). Previously proposed models (9, 14, 16) suggested that the open versus closed conformations of UvrD correspond to moving versus stalled states; however, our direct observations demonstrate that these conformations instead correlate with UvrD rezipping versus unwinding activity. Plotting the average speed versus FRET efficiency for many individual FRET-determined time intervals (Fig. 3D) corroborates our finding that unwinding (positive velocity) and rezipping (negative velocity) correspond to high and low FRET states, respectively. Dividing the data traces into time intervals by unwinding speed instead yields similar results (fig. S5).

A simple model explains the correlation between 2B domain orientation and UvrD unwinding and rezipping activity (Fig. 3A and movie S1) (19). In the closed conformation, the 1A-2A motor domains are oriented such that 3′-to-5′ translocation occurs toward the duplex, in the unwinding direction (14). In the transition from closed to open, the 2B and 1A-2A domains rotate ~150° relative to each other (16), consistent with our FRET efficiencies. We propose that an unwinding-to-rezipping reversal is initiated by the 1A-2A domains disengaging from the ssDNA strand while 2B anchors UvrD at the DNA junction, preventing its complete dissociation. A switch from closed to open, along with rotation of 2B about the duplex, then reorients the 1A-2A domains to bind the opposing ssDNA strand in the proper orientation. 3′-to-5′ translocation is now directed away from the fork junction, allowing the duplex to rezip. These steps are reversible, leading to robust back-and-forth switching between unwinding and rezipping activities. Our model is compatible with previous studies of UvrD (6) and its SF 1 homologs (19, 22, 23) and with the observation that intramolecular cross-linking of a Rep or PcrA monomer into the closed form allows it to unwind >1000 bp processively (27).

Although our model provides a mechanism for monomer reversals, it does not explain why these occur in the first place or why they occur less frequently during dimer unwinding. Additional measurements varying duplex stability indicate that reversals occur whenever UvrD encounters an energetic barrier to unwinding (19). UvrD dimers (and higher-order assemblies) may be more likely to overcome this barrier, leading to long-distance unwinding. Our data, together with past mutational studies (6, 28), point to strand-switching being the primary inhibitor of long-distance unwinding. For dimers, direct contact between helicases may inhibit strand-switching by the lagging UvrD, either by applying force to the leading UvrD, which stabilizes it at the duplex junction, or by preventing 2B interactions with the duplex DNA.

It is plausible that these two levels of unwinding activity play biological roles. UvrD is involved in multiple, distinct DNA maintenance processes that require different levels of processivity. During nucleotide excision repair, it is estimated that only ~15 bp of DNA are unwound (29). The strand-switching model above may provide a mechanism by which UvrD can unwind a small number of base pairs yet remain engaged with the DNA near the site of damage. In contrast, methyl-directed mismatch repair can require more than 1000 bp of DNA to be unwound (30). UvrD conformation and stoichiometry may be critical in enabling and regulating these disparate functions. Interactions of UvrD with accessory proteins, such as MutL (2), may also influence its conformation, stoichiometry, and activities.

Supplementary Materials

www.sciencemag.org/content/348/6232/352/suppl/DC1

Materials and Methods

Figs. S1 to S8

References (3142)

Movie S1

References and Notes

  1. See materials and methods and other supplementary materials on Science Online.
  2. Acknowledgments: We thank members of the Chemla (including Z. Qi), Ha (including K.-S. Lee), and Lohman (including E. Tomko) laboratories for scientific discussion and assistance in the experimental design. Funding was provided by NSF grants MCB-0952442 (CAREER to Y.R.C.) and PHY-1430124 (Center for the Physics of Living Cells to Y.R.C. and T.H.); NIH grants R21 RR025341 (to Y.R.C. and T.H.), R01 GM065367 (to T.H.), and R01 GM045948 (to T.M.L.); and the Alfred P. Sloan Research Fellowship (to Y.R.C.). T.H. is an investigator with the Howard Hughes Medical Institute.
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