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Originally published In Press as doi:10.1074/jbc.M407193200 on September 22, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54529-54532, December 24, 2004
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Closing of the Fingers Domain Generates Motor Forces in the HIV Reverse Transcriptase*

Hailong Lu, Jed Macosko{ddagger}, Diana Habel-Rodriguez, Rebecca W. Keller, James A. Brozik, and David J. Keller§

From the Department of Chemistry, The University of New Mexico, Albuquerque, New Mexico 87106

Received for publication, June 28, 2004 , and in revised form, September 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (~15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human immunodeficiency virus reverse transcriptase (HIV1 RT) is the enzyme responsible for copying the RNA genome of the AIDS virus into a double-stranded DNA provirus, which is incorporated into the host genome. It has three distinct functions: 1) polymerizing a DNA strand complementary to the genomic RNA, thus forming an RNA-DNA hybrid (reverse transcriptase activity); 2) degrading the original RNA, leaving behind a single-stranded DNA copy (RNase H activity); 3) generating a second DNA strand complementary to the first strand, creating the double-stranded DNA provirus (DNA polymerase activity) (1). Because creating the provirus is a necessary central step in HIV infection, HIV RT is the target of many anti-AIDS therapeutic drugs such as azidothymidine, lamivudine, efavirenz, and nevirapine (2–4). More generally, the structure and catalytic mechanisms of HIV are closely related to those of other well studied reverse transcriptases (e.g. MMLuV) (5) and small DNA polymerases (e.g. T7 DNA polymerase, Escherichia coli Pol I) (6–8). Hence, understanding how HIV RT works, how its functional capabilities arise from structure and sequence, is important both for medicine and for basic science.

During one polymerization cycle, any polymerase must align the primer-template DNA with the catalytic site, orient the downstream template base, bind and align the incoming nucleoside triphosphate (NTP) monomer, recognize the complementary NTP and reject others, catalyze the formation of the new phosphodiester bond, release the pyrophosphate product, and finally move along the DNA to the next location. Except for the bond-forming reactions, all of these steps can be viewed as mechanical processes made possible by motions and interactions among protein domains acting together like parts in a protein machine. A number of recent crystal structures (9–11) have elucidated the nature of the key parts and motions (see Fig. 1a), and ensemble kinetics measurements have helped define the main biochemical states (12–14) (see Fig. 1b), but to date no mechanical studies of HIV RT have been reported. In part this is because of the difficulty of measuring motor-like forces and motions of proteins, which tend to average out in ensembles and so must be measured on individual molecules. HIV RT presents a particular challenge because its processivity is low (for DNA polymerase activity, ~20 base pairs) (14, 15) and its step size is small (~0.34 nm/base incorporated), so the net movement along the DNA during a single polymerization run is small.



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  		FIG. 1.
a, open (Protein Data Bank entry 2HMI [PDB] ) and closed (Protein Data Bank entry 1RTD [PDB] ) structures of HIV RT. The three major domains of HIV RT are colored green (fingers), red (palm), and blue (thumb). When the polymerase changes from the open to the closed state the fingers domain clamps down on the DNA at the junction between the single-stranded template region and the double-stranded primer-template region. Small movements of the thumb and palm also occur. b, a minimal kinetic mechanism for processive DNA polymerization (17). Beginning in the open state without bound nucleotide, the polymerase binds nucleotide, closes to form a tight catalytic complex, catalyzes the formation of a new phosphodiester bond, opens, and releases pyrophosphate to return to the initial state. At some point in the turnover cycle motor forces are generated, and at some point (not necessarily the same) the polymerase moves to the next position on the template.

 
To overcome this problem, we have developed a single molecule motor force and motion experiment using an atomic force microscope cantilever as the force sensor. The atomic force microscope has the spatial resolution to detect short runs of polymerization by HIV RT and sufficient force resolution to detect its motor forces. By measuring polymerization velocity under increasing load until the motor stalls, the experiments have been used to construct an approximate force-velocity curve. The curve shows a stall force of about 15 pN for HIV RT, and the shape of the curve is consistent with the load force acting strongly on the rate-limiting step of the polymerase turnover cycle. These results, together with ensemble kinetics and structural information, suggest that the closing of the fingers domain is a major force-generating step for the HIV RT molecular motor.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Biotinylated primer-template DNA (Fig. 2b) was formed by annealing the primer to a 5'-biotinylated template oligonucleotide (both purchased from Operon (Alameda, CA) from 70 to 21 °C. HIV RT was purchased from Worthington (Lakewood, NJ) or Amersham Biosciences. Biotin-bovine serum albumin was purchased from Sigma. Neutravidin, which is thought to have less nonspecific binding than streptavidin, was purchased from Pierce. All motor force experiments were carried out with a modified NanoScope IIIa atomic force microscope controlled externally by a computer equipped with DAQ boards and running software written in LabView (National Instruments, Austin, TX). Triangular cantilevers with force constant 10 pN/nm (Digital Instruments, Santa Barbara, CA) were used. To reduce thermal drift without loss of detector sensitivity, the gold coating was stripped from the cantilever arms and base but retained near the apex of the triangle by dipping the tip end in rubber cement, dissolving the gold with HCl/HNO3, and removing the excess rubber cement with chloroform. Tips were then cleaned by extensive ozonolysis and UV irradiation. HIV RT was adsorbed to tips by placing a 5-µl 0.5 µM droplet on the tip for 3 min and then rinsing with 1 ml of buffer A (10 mM MgCl2, 40 mM KCl, 25 mM Tris·HCl (pH 7.8)). DNA was attached to the cover glass in a 3-step process: a 10-µl droplet of 1.8 mg/ml biotinylated bovine serum albumin placed on the glass for 5 min, followed by a rinse with buffer A; a 10-µl droplet of 1 mg/ml neutravidin for 5 min, followed by a second rinse; and finally a 10-µl droplet of 1 µM biotinylated primer-template DNA for 5 min and a final rinse.



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  		FIG. 2.
a, schematic of the motor-force experiment. b, primer-template DNA used in the motor force experiments. HIV RT is adsorbed to the atomic force microscope tip (~20-nm end diameter), and short primer-template DNA is attached to the glass substrate by biotin-neutravidin-bovine serum albumin. A saturating concentration of all four nucleotides is present in solution.

 
Control experiments were carried out by omitting either the primertemplate DNA or the nucleotides but were otherwise identical to full experiments. Control experiments were typically very clean with little thermal drift and no binding between tip and surface. The root mean square thermal noise observed was usually less than 0.2 nm (2 pN) in a 10-Hz bandwidth. After repeated physical contacts between tip and surface (30–40 cycles, see below), sticking and stretching of polymeric material between tip and surface was sometimes observed. We attributed this to denatured protein on the tip and usually stopped the experiment after ~20 contacts or whenever sticking occurred.

The force-velocity curve was constructed by spline-fitting experimental traces of cantilever deflection, x, versus time and then calculating the number of bases polymerized at each time using the freely jointed chain model without intrinsic stretch modulus (16), n(t) = (h - F(t)/{kappa})/(lL(F(t)l/kBT)), where h = F(0)/{kappa} + n(0)lL(F(0)l/kBT), F(t)) is the force of cantilever deflection at time t, {kappa} is the cantilever force constant (10 pN/nm), l is the length of one single-stranded monomer unit (0.7 nm in our calculations), kB is Boltzmann's constant, and T is temperature. The function L(x) is the Langevin function, L(x) = 1/tanh(x) - 1/x, and F(0) and n(0) are the initial force and DNA template length (in bases), respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Motor Forces Versus Time—The basic motor force experiment is illustrated in Fig. 2a. HIV RT is adsorbed to the sharp tip of the cantilever (20–50 nm radius), which allows the polymerase to approach the surface to within a few nanometers without unwanted contacts. A 5'-biotinylated, 56-base DNA template with a 10-base primer annealed at its 3'-end is attached to a glass surface by a layer of neutravidin bound to a layer of biotinylated bovine serum albumin. The substrate is mounted on a piezoelectric positioner and can be raised or lowered to bring the DNA closer or farther from the tip. Finally, a saturating concentration (100 µM) in each of the four nucleotides in the surrounding buffer ensures that once a "hookup" takes place, the polymerase molecule will fill in the template, shortening the DNA tether and deflecting the cantilever.

The results of several experiments are shown in Fig. 3, a–c. At the beginning of each experiment the substrate is raised (top trace) until the tip touches lightly (~20 pN), establishing the tip-surface distance. The substrate then retracts to break any hookups that may have occurred during the touch and then reapproaches, stopping at a preset tip-sample distance, (8–15 nm). Calculations with freely jointed chain or worm-like chain models for the DNA suggest that at this range of distances the free ends of the DNA will bind to the tip-adsorbed polymerase molecules at a reasonable rate (between ~0.1 and 5 s-1). The tip hovers above the substrate for a time (~10 s), "fishing" for hookups and recording the resulting cantilever deflections. Finally the surface retracts again at the end of the experimental cycle. At the end of each cycle the tip is moved laterally (by about 20 nm) so that each cycle interrogates a new spot on the substrate surface. Motor events occurred only about once every several hundred cycles.



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  		FIG. 3.
Force versus time for HIV RT polymerization. The top trace in each graph shows movement of the piezoelectric positioner in nanometers. a, a polymerization run followed by DNA stretching (in blue circle). b, a polymerization run showing a hookup transient. c, a slow and a fast polymerization run. The fast run shows features that are consistent with single base steps of DNA

 
In Fig. 3a, polymerization begins rapidly (~10.5 s) from the initial approach, but as load force builds up (~13 pN) the polymerase slows to a near stall. On this occasion the sample retracts from the tip while the hookup is still intact, so the DNA is mechanically stretched. At ~40 pN the hookup breaks, either because the DNA dissociates from the HIV RT or some other link between tip and substrate breaks.

Fig. 3b shows two traces, one in which no hookup occurred (middle curve, essentially identical to one of the traces in a control experiment) and one taken immediately afterward (bottom curve) that is similar to the trace in Fig. 3a except that dissociation occurs without mechanical stretching. This trace reveals a "hookup transient", a small, sudden force preceding the main run, which is fairly frequently seen (the curve in Fig. 3a may also have one). We have interpreted these transients as binding between DNA and HIV RT under conditions where the DNA is slightly stretched. This run also demonstrated that the polymerase continues to "stutter" slowly forward even after a near stall (~10 pN). HIV RT has no (proofreading) 3'-5'-exonuclease activity and, in the absence of pyrophosphate, is unable to reverse polymerization by pyrophosphorolysis. Polymerization is thus effectively irreversible, meaning that the motor can move slowly forward under high load force by taking advantage of favorable thermal fluctuations (17).

Fig. 3c illustrates the variety expected of stochastic process; one run is slow and steady, the immediately following run is fast and erratic. Interestingly, the stuttering phase of the lower curve seems to show a series of three roughly flat regions separated by ~3.5 pN, which corresponds to the expected net cantilever deflection for incorporation of one base under the prevailing template tension. Though the noise is too large to make a clear identification, these features (and those observed in other runs) are consistent with single base steps of DNA replication.

Force-Velocity Curve—Each motor force event in Fig. 3 reveals the velocity of the polymerase motor over a full range of applied load forces, from near zero to stall. Fifteen such events have been used to construct a rough force-velocity curve (Fig. 4). The polymerase velocity at zero load was measured earlier using single molecule fluorescence methods.2



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  		FIG. 4.
Force-velocity data for HIV RT. Inset, a log plot of the graph. 90% of the data fall into the region between the red and blue dashed lines, which correspond to movements of 30 and 10 Å, respectively. The green line corresponds to a movement of 16 Å.

 

The shape of the force-velocity curve provides two important pieces of information. First, the velocity drops immediately and rapidly, even at low load. External forces can only affect mechanical processes, i.e. transitions that involve a significant physical motion along the direction in which the force is applied. If the force were acting only on a fast kinetic step in the turnover cycle, the velocity would be only slightly affected by load and the force-velocity curve would be nearly flat at low load. Only at high force, when the affected step has been slowed until it has become rate-limiting, would the velocity decrease rapidly. Thus, the fact that the velocity falls rapidly even at the smallest forces implies that the load is acting on the "natural" (zero load) rate-limiting step and that this step is mechanical in nature. Previous work has identified the closing of the fingers domain to form a tight catalytic complex with incoming nucleotide as the zero-load rate-limiting step (18). This is indeed a mechanical step, consistent with our results. Second, at forces near stall the velocity must be approximately proportional to the rate-limiting rate constant (at that force), and the shape of the curve therefore gives the dependence of this rate constant on load force. Thus, we take the velocity in this region to have an Arrhenius form, v = v0e -({Delta}H0 - F{Delta}x)/kBT, where {Delta}x is the effective size of the motion associated with the transition. A plot of log velocity versus force should then be linear with slope proportional to {Delta}x. As shown in Fig. 4, our data are consistent with a motion of between 1 and 3 nm, with 1.6 nm the most likely value. This is too large to be fingers movement alone (~1 nm or less for HIV RT, as measured from crystal structures in open and closed states (9)) and so must involve a combination of steps, including (possibly) template base stacking (up to ~0.7 nm) and translocation (up to 0.34 nm). Nonetheless, both the low force and high force regions of the force curve are consistent with the idea that load force acts on the fingers-closing step and that fingers-closing is probably the main force-generating process for the HIV RT molecular motor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ability to generate force is crucial to the function of all DNA and RNA polymerases; movement along the DNA, displacement of DNA-bound protein and non-template DNA strands, clearing loops and stems in the template, and negotiating nucleosomes all require force. The combination of motor force measurements and single molecule fluorescence points toward the fingers-closing step as a major force-generating transition within the overall polymerization cycle. A similar conclusion was reached from previous measurements on the motor forces of T7 DNA polymerase (19), which, like HIV RT, is the replicase for its respective organism and seems to function by the same basic mechanism. The fingers domain thus appears to play several key roles in the functioning of the polymerase machine, including clamping the template base, incoming nucleotide, and catalytic site in the correct relative orientations; helping to recognize the complementary nucleotide and reject mismatches; coordinating the various steps (nucleotide binding, catalysis, translocation) of the turnover cycle; and, in light of the data presented here, generating motor forces. The fingers domain and its motions thus emerge as perhaps the defining feature of all polymerases.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant GM63808 (to D. J. K. and J. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a Discovery Institute research fellowship. Back

§ To whom correspondence should be addressed. Tel.: 505-277-1653; Fax: 505-277-2609; E-mail: dkeller{at}unm.edu.

1 The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; pN, piconewton. Back

2 T. P. Ortiz, J. A. Marshall, L. A. Meyer, R. W. Davis, J. C. Macosko, D. J. Keller, and J. A. Brozik, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Basame Solomon, Dr. Jason A. Marshall, Jeremy Shelton, and Theodore P. Ortiz for help.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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This Article
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