©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Phage T4-coded DNA Replication Helicase (gp41) Forms a Hexamer upon Activation by Nucleoside Triphosphate (*)

(Received for publication, November 22, 1994; and in revised form, January 16, 1995)

Feng Dong (1) Edward P. Gogol (1) (2) Peter H. von Hippel (1)(§)

From the  (1)Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229 and the (2)Division of Cell Biology and Biophysics, University of Missouri, Kansas City, Missouri 64110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of 10^6M. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATPS and GTPS and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.


INTRODUCTION

Studies of a variety of systems that catalyze the elongation phase of DNA replication (Kornberg and Baker, 1992) have shown that most can be described in terms of three functional components or subassemblies. These are: (i) the central polymerase molecules, which often carry a 3` 5` exonuclease (editing) activity in addition to their template-directed 5` 3` polymerization function; (ii) a set of accessory proteins that interact with the polymerases to form ``holoenzyme'' complexes that can synthesize leading strand DNA processively and at physiological rates and fidelities; and (iii) helicase-primase (``primosome'') complexes that open the double-stranded DNA ahead of the overall replication complex, prime (with RNA) the initiation of the discontinuously synthesized lagging strands, and integrate the function of the holoenzymes involved in leading and lagging strand synthesis with the overall movement of the replication fork. The number of discrete protein subunits required to operate and integrate each of these functional subassemblies varies from one organism to another, but this general pattern of organization seems to prevail from simple bacteriophages to complex eukaryotes.

In keeping with this organizational pattern, the elongation phase of DNA replication in bacteriophage T4 also requires the coordinated interaction of three such sets of components, which here consist of seven discrete T4-coded protein species (for reviews, see Alberts (1987), Cha and Alberts(1988), or Nossal(1994)). These are the DNA-dependent DNA polymerase (gp43), (^1)which together with three accessory proteins (gp44, gp45, and gp62), and a single-stranded DNA-binding protein (gp32), form the processive holoenzyme. Efficient DNA replication on double-stranded DNA templates also requires the participation of a primosome subassembly, which in T4 contains helicase (gp41) and primase (gp61) subunits and catalyzes the ATP or GTP-driven unwinding of the double-stranded DNA template, as well as the synthesis of the pentameric RNA primers required for coordinate lagging-strand DNA synthesis. This relative parsimony of required components has made the T4 system a useful paradigm system for mechanistic studies of DNA replication.

The interactions of the polymerase and the accessory proteins within the T4 holoenzyme are reasonably well understood (see Young et al. (1992, 1994a) for recent reviews), but little is yet known about the properties and function of the primosome at the structural or mechanistic level. Many lines of evidence suggest that gp41 and gp61 interact to form a primosome complex that provides both primase and helicase activities. In studies in particular from the Alberts and Nossal laboratories, gp41 and gp61 have been purified and characterized as enzymes, and some aspects of their helicase and primase functions have been described. The gp41 helicase exhibits a single-stranded DNA-stimulated GTPase/ATPase activity and a 5` 3` DNA helicase activity that is driven by PuTP hydrolysis (Morris et al., 1979; Liu and Alberts, 1981a; Venkatesan et al., 1982). Analysis of the dependence of the steady-state kinetics of this DNA-stimulated PuTPase activity as a function of single-stranded DNA length has also shown that PuTP hydrolysis drives processive (and unidirectional) gp41 translocation along the DNA (Young et al., 1994b, 1994c; Liu and Alberts, 1981a).

The gp61 primase alone can catalyze limited, templatedependent RNA primer synthesis, with the dimers ppApC and ppGpC as major products (Hinton and Nossal, 1987; Nossal and Hinton, 1987; Cha and Alberts, 1990). However, the physiologically relevant primers produced by the T4 primosome are pentaribonucleotides (Kurosawa and Okazaki, 1979), and the dinucleotides made by gp61 primase alone are utilized only poorly by the T4 DNA polymerase as primers of lagging-strand DNA synthesis.

It is clear that the physiologically relevant properties and activities of gp41 and gp61 are only displayed when these two proteins are integrated as a primosome complex. Thus the primosome is required to unwind double-stranded DNA at high rates, to synthesize the physiologically relevant pentaribonucleotide RNA primers efficiently (Cha and Alberts, 1986; Hinton and Nossal, 1987; Liu and Alberts, 1980, 1981b), and to coordinate leading and lagging strand DNA synthesis at a DNA replication fork (Nossal and Hinton, 1987; Cha and Alberts, 1990).

As a step in developing a molecular picture of the proteins of the T4 primosome, we present here a detailed study of the assembly and properties of gp41 alone. We show that at physiological protein concentrations this protein exists primarily as a dimer, although at somewhat lower concentrations a dynamic monomer dimer equilibrium can be demonstrated. On binding ATP or GTP substrates (or their nonhydrolyzable analogs), these dimers assemble to a hexameric complex that can interact with DNA models of the replication fork and exhibit the DNA-stimulated PuTPase activity required for helicase action. Cross-linking and cryoelectron microscopic studies suggest that the hexamer is structured as a trimer of asymmetric dimers in the form of a ring-shaped hexagon. Under ``Discussion,'' this complex is compared with those of other (largely hexameric) helicases that have recently been characterized, and the functional implications of this assembly mode are considered.


MATERIALS AND METHODS

Reagents and Buffers

The protein cross-linking reagent dithiobis(succinimidyl propionate) (DSP) was purchased from Sigma. Chemicals for buffers, for gel electrophoresis (including acrylamide, bisacrylamide, TEMED, and ammonium persulfate), and for silver staining protein gels were all of gel electrophoresis or analytical reagent grade. Purine nucleotide triphosphates and their non-hydrolyzable analogs were obtained from Boehringer Mannheim. A number of buffers were used in this study. They are listed here, together with the abbreviations that have been used to designate them in the text. HEAC (used in gp41 purification) consisted of 10 mM HEPES, 25 mM KOAc, 2 mM Mg(OAc)(2), 0.1 mM EDTA, 1 mM DTT, and 25% glycerol, pH 7.5. HEAC-plus (used for storing gp41 unfrozen at -20 °C) was the same as HEAC buffer, but contained 50% glycerol (instead of 25%). HKAE (used for oligonucleotide hybridization) consisted of 50 mM HEPES, 100 mM KOAc, 1 mM EDTA, pH 7.6. TAMK (used in analytical ultracentrifugation, protein cross-linking, and cryoelectron microscopy experiments) consisted of 33 mM Tris-OAc, 6 mM Mg(OAc)(2), 50 mM KOAc, 1 mM DTT, pH 7.8 (this buffer is very similar to that used by Liu and Alberts (1981a) except that bovine serum albumin was omitted since it interferes with physical biochemical measurements). TAMK-plus (used in HPLC exclusion chromatography) was the same as TAMK but contained 150 mM KOAc instead of 50 mM. For composition of TAE and TBE (gel electrophoresis running buffer), see Maniatis(1982). TCES (used in gp41 purification) consisted of 50 mM Tris-HCl, 1 mM EDTA, 20% sucrose, pH 7.5. TE consisted of 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. TE(low) consisted of 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5.

Protein Purification and Properties

The T4-coded replication helicase (gp41) has been cloned and overexpressed by Nossal and co-workers (Hinton et al., 1985). Large scale preparation and purification of gp41 was achieved by overexpressing the protein in OR1265 Escherichia coli cells containing plasmid pDH518 (kindly supplied by Drs. Hinton and Nossal), using a slightly modified version (^2)of the procedure of Richardson and Nossal (1989b). Cells were grown in a 30-liter fermentor under the conditions used by Hinton et al.(1985). Cells were harvested by centrifugation and were stored at -80 °C until use. All subsequent steps were carried out at 0 °C (on ice) unless otherwise specified. Each purification was done using a few grams of cell paste and was started with suspending the cell paste using 20 ml of TCES buffer for each gram of cell paste. Cells were lysed at 15,000-16,000 p.s.i. by two passages through a French Pressure cell press (American Inst. Co.) that had been precooled in a 4 °C coldroom. After cell lysis the solution was made 20 mM in Mg(OAc)(2) by addition of 1 M Mg(OAc)(2) solution, and centrifuged in a Beckman 60 Ti rotor for 2 h at 40,000 rpm (4 °C). The supernatant was recovered and mixed with an equal volume of 32% (NH(4))(2)SO(4) in HEAC buffer to precipitate the protein. After letting the solution stand overnight at 4 °C with slow stirring, the precipitate was removed by centrifugation at 17,000 rpm in a Beckman 60 Ti rotor at 4 °C, and the pellet was rinsed briefly with a solution containing 16% (NH(4))(2)SO(4) in HEAC buffer and then redissolved in HEAC buffer (2 ml of buffer for each gram of cell paste used at start up). This ammonium sulfate precipitation step was repeated a second time, and the resulting protein pellet was redissolved in HEAC buffer (4 ml for each gram of cell used at start of purification). After the nonsoluble material was removed by centrifugation in a microcentrifuge for 5 min at 4 °C, the solution was dialyzed at 4 °C against three or four 250-ml volumes of HEAC-plus buffer over a period of 24 h in Spectro/Por3 (M(r) cut-off: 3500) dialysis tubing (Spectrum Medical Industries, Inc.). The resulting protein stock solutions were stored at -20 °C. The protein stains with Coomassie Blue G250 as a single band after SDS-polyacrylamide gel electrophoresis, but additional minor bands can be visualized in silver-stained gels. Concentrations of gp41 protein were determined by UV absorbance at 280 nm using a molar extinction coefficient () of 7.6 times 10^4M cm, calculated as described by Young et al. (1994c).

Preparation of Synthetic DNA Replication Fork Constructs

Many of the protein-protein cross-linking and cryoEM experiments reported here were carried out in the presence of a synthetic DNA replication fork construct consisting of two partially complementary DNA oligonucleotide sequences (53-mer and 48-mer, respectively). These strands were hybridized together to form an 18-base pair double-stranded DNA ``handle'' and two single-stranded DNA ``tines,'' as shown in Fig. 1A. Some cross-linking studies were also performed using the construct shown in Fig. 1B. The component oligonucleotides of both fork constructs were synthesized in the University of Oregon Biotechnology Laboratory and by Midland Certified Reagent Co. (Midland, TX) and were purified by preparative electrophoresis on denaturing polyacrylamide gels (Maniatis et al., 1982b), followed by elution as described by Maniatis et al. (1982a). The oligonucleotides were further purified by HPLC using a Beckman Ultrapore C4 column, lyophilized, redissolved in TE(low) buffer, and stored at -20 °C. The concentration of each purified oligonucleotide was determined by UV absorbance, using extinction coefficients calculated by the nearest-neighbor method as described by Warshaw and Cantor(1970). The molar extinction coefficients () for the 53-mer and the 48-mer DNA in dilute aqueous salt solution were 4.3 times 10^5 and 4.0 times 10^5M cm, respectively.


Figure 1: Nucleotide sequences of the DNA replication fork constructs that were used as the helicase binding substrate in the protein-protein chemical cross-linking and the cryoelectron microscopic imaging experiments. A, a forked DNA template with a double-stranded DNA handle and two single-stranded DNA tines. The lengths of the two single-stranded tines on this forked DNA template (30 and 35 nucleotides for the 3` and 5` overhanging single-stranded sequences, respectively) were designed on the basis of the known requirements of the helicase (Venkatesan et al., 1982; Richardson and Nossal, 1989a; Hinton et al., 1987), and the two single stranded homo-oligonucleotides were used to minimize the formation of possible secondary structures. B, a forked DNA template that has been used in our laboratory for studies of the T4 DNA replication accessory proteins (Reddy et al., 1993).



The synthetic fork constructs were assembled from the component strands by mixing equimolar quantities of the partially complimentary oligonucleotides in HKAE hybridization buffer, usually at 10-100 µM concentrations. Samples were incubated at 80 °C for 2-5 min to remove residual secondary structure and then slow-cooled to room temperature over a period of 1-2 h to allow hybridization to proceed. The formation of unique hybrid complexes was assayed and verified by electrophoresis on a 10% (20:1 acrylamide:bisacrylamide) nondenaturing polyacrylamide gel. Gels were run at low current (20 mA) in TAE running buffer to prevent heating, and 5 mM MgCl(2) was added to both the gel and the running buffer to stabilize the hybridized complexes. Aliquots of the synthetic fork constructs were stored frozen at -20 °C in HKAE buffer.

Analytical Ultracentrifugation

Sedimentation velocity experiments were performed in the van Holde laboratory at Oregon State University, using a Beckman model XLA analytical ultracentrifuge. All sedimentation experiments were carried out in 1.2-cm pathlength double-sector cells with quartz windows. Gp41 concentrations were set at 5 µM (in monomer units) in TAMK buffer (which also contained 12.5% glycerol carried over from the HEAC-plus buffer used for gp41 storage) and were measured at 280 nm using the UV scanner. When present the concentrations of PuTP analogs (usually GTPS) were 0.1 mM. Sedimentation velocity experiments were carried out at temperatures between 20 and 25 °C in either a two-hole AN-D rotor or a four-hole AN-F rotor at 50,000 rpm, and the data were analyzed by the method of van Holde and Weischet(1978).

Hydrodynamic Calculations

Calculation of the sedimentation coefficient of the gp41 protein monomer was carried out as described by van Holde(1975). The sedimentation coefficient of a macromolecule (s^0) at low protein concentrations is a function of its size, shape, and density, as well as of the properties of the solvent. Thus,

where M is the molar mass and v the partial specific volume of the molecule, is the density of the solvent, f is the frictional coefficient, and N(0) is Avogadro's number. For an unhydrated, spherical macromolecule

where is the viscosity of the solution and R(0) the radius of the molecule. Hence, from ,

Therefore, for an unhydrated and spherical molecule,

when s^0 is in Svedbergs and all other factors are in cgs units.

In our calculations we have modeled the gp41 monomer as a hydrated spherical protein, using the empirical equation noted by van Holde (1975), which has been shown to be applicable to many globular proteins of average hydration

Here s(1)^0 is the sedimentation coefficient (in Svedbergs) of the gp41 monomer at 20 °C in water. The molar mass and partial specific volume for the gp41 protein were calculated on the basis of the amino acid composition of the protein, and were 53,554 g/mol and 0.735 cm^3/g, respectively.

Sedimentation coefficients for the gp41 dimer and various hexamer forms were calculated using the hydrodynamic simulation method ofKirkwood(1954) and the equation of van Holde(1975):

and

where s is the sedimentation coefficient predicted for a rigid structure containing n identical spherical beads, each with radius R and sedimentation coefficient s. R is the center-to-center distance between these beads. Therefore each value of alpha corresponds to a ratio of an intersubunit distance to the subunit radius that has been derived on the basis of the assumed geometry of the complex.

As a consequence the sedimentation coefficient for any complex can be calculated using:

HPLC Experiments

High performance molecular size exclusion chromatography was used to study the gp41 monomer ⇔ dimer equilibrium at low (submicromolar) gp41 concentrations. A GF450 gel filtration HPLC column (Zorbax Bio-Series, Du Pont), suitable for separating large protein complexes, was used with a Beckman Gold HPLC System (consisting of a Beckman Programmable Pump Module 126, a NEC PC-8300 controller, a Beckman Programmable Detector Module 166, and a Spectra-Physics SP4279 Integrator). The dimensions of the GF450 column were 9.4 mm (inner diameter) times 25 cm (length). The system was equilibrated with TAMK-plus buffer by washing with more than 20 column volumes. Sample injections were performed at various concentrations of gp41 in TAMK-plus buffer and at volumes ranging from 25 to 100 µl. Elutions were carried out at a constant flow rate of 0.5 ml/min, and protein elution profiles were monitored by UV absorbance at 280 nm. The total peak area corresponding to the eluted protein was normalized to the total amount of protein injected for each chromatogram, and total protein concentrations in monomer units in the eluted samples were then calculated directly from elution volumes. The concentrations of each protein species were then estimated on the basis of the fraction of the total peak area corresponding to each.

Association constants (K) for the gp41 dimerization reaction were estimated using:

Here f(m) represents the fraction of monomer present at each total gp41 concentration ([gp41]). The data were plotted as f(m)^2/(1 - f(m)) versus (1/[gp41]), and K was calculated from the slope of the best straight line fitted to the data with an intercept of zero.

Protein-Protein Chemical Cross-linking with DSP

Protein-protein cross-linking experiments with DSP were performed using a modified version of the procedure described by Finger and Richardson(1982). All cross-linking experiments were performed in TAMK buffer without DTT (DTT was omitted to avoid cleaving the disulfide bond of the DSP cross-linker). Purine nucleotide triphosphates (PuTP), non-hydrolyzable PuTP analogs (PuTPS), and DNA replication fork constructs were included in some experiments. Generally PuTP and PuTPS were added to final concentrations of 1 mM, and final gp41 concentrations were 200 nM, 1 µM, and 5 µM (in protein monomers). Synthetic DNA fork constructs were added to final molar concentrations equal to the total gp41 concentration (as moles of monomers) present in each experiment. After mixing and incubation at room temperature for 1 min, cross-linking was started by adding small amounts (usually 2-3% of the total reaction volume) of freshly prepared 5% (w/v) DSP stock solution in dimethylformamide (0.5% DSP stock solutions were used in some experiments, as indicated). After incubation for various times at room temperature, aliquots were removed from the reaction mixture and the cross-linking reaction was quenched by adding 0.1 volume of a 1.4 M ethanolamine-HCl (pH 8.0). The samples were held at room temperature for 20 min, each aliquot was made 1% in SDS and 10% in glycerol (by volume), incubated at 37 °C for 30 min, and analyzed by SDS-polyacrylamide gel electrophoresis, using either the discontinuous gel electrophoresis system of Laemmli(1970) or the phosphate-SDS gel system of Weber and Osborn(1969, 1975). The gels were stained with silver.

Cryoelectron Microscopy

Samples of gp41 (in the presence and absence of nucleotides and/or of forked DNA constructs) were prepared for cryoelectron microscopy using concentration and buffer conditions similar to those used for cross-linking. Stock solutions of gp41 were diluted to concentrations ranging from 0.1 to 5 µM in HP buffer shortly before use. When present, forked DNA constructs were added to final molar concentrations equivalent to one-half to one-fifth of the total concentration of gp41 (as monomers). Solutions were incubated at 37 °C for 2-5 min prior to addition of nucleoside triphosphates. ATP and GTP (or the ATPS and GTPS) were added at concentrations of 1 or 2 mM, and the solutions were held at 37 °C for 1 min (unless stated otherwise) prior to cryoEM sample preparation.

Specimens were applied to EM grids coated with perforated carbon films, and were blotted and frozen as described elsewhere (Gogol et al., 1992). Sample preparation was carried out in a 4 °C coldroom to minimize evaporation prior to freezing. Solutions containing gp41 were maintained at 37 °C from the time of nucleotide addition until applied to the grids. Three or four samples were prepared from each solution within approximately 1 min, resulting in a standard incubation time (at 37 °C) in the presence of nucleotide triphosphates of 1.2-2 min between the freezing of the first and last grids. Grids were subsequently transferred and stored under liquid nitrogen until examined in the cryoelectron microscope.

The specimens were imaged using a Philips EM400 electron microscope equipped with a low dose operations kit (Philips Electronic Instruments, Mahwah, NJ) and a custom-made double-bladed anticontaminator. Specimens were loaded in a Gatan (Pleasanton, CA) cryotransfer apparatus and maintained at temperatures below -160 °C at all times. Images were recorded on Kodak SO163 film using minimal-dose (20 e/A^2) methods at a magnification of 57,000. The magnification of the microscope was calibrated by imaging thin negatively stained crystals of catalase at the same objective lens current used to record the gp41 images. Images were recorded at a defocus of 1 µm, which correctly represents spatial information down to a resolution of approximately 20 A.

Image alignment and averaging were performed with the SPIDER software package (Frank et al., 1981). Areas of micrographs, selected for image clarity and the presence of a suitable number of particles, were digitized with an Eikonix 1412 digitizing camera at a magnification that yielded individual 25 times 25-µm image elements on the film, corresponding to 4.4 times 4.4-A areas on the specimen. Digitized images were displayed on a VaxStation 3100 (Digital Equipment Corp., Woburn, MA), and individual particles were selected from the regions of thin ice that spanned the holes in the carbon support film.

The only criterion used to select molecules for image analysis was that the chosen particles be fairly circular in shape and show no obvious overlap with nearby molecules. Images were aligned by iterative translational and rotational matching to a reference particle chosen from the micrograph on the basis of its clarity and contrast. Subsequent cycles of alignment used the averaged image as a reference. Different starting images were used to test the dependence of the final alignment and the resulting averaged image on the choice of initial reference image, and the results of these parallel alignments were compared visually. In most cases, alignments starting with different reference images converged to visually identical averaged images after the second cycle. For the most highly variable data set (the nucleotide-gp41 complexes in the absence of DNA), a ``reference-free'' alignment procedure described by Penczek et al.(1992) was applied. This procedure reduced the dependence of the final average on the selection of the initial reference. For each set of images 10% of the aligned images were discarded on the basis of perceived poor alignment (judged both visually and by correlation coefficient versus the reference image), and the remainder were averaged.


RESULTS

States of Association (Oligomerization) of the Gp41 Helicase

Liu and Alberts (1981a) used a sucrose gradient sedimentation method to examine the states of association of gp41 and showed that the apparent sedimentation coefficient of gp41 increased in the presence of 1 mM GTPS. They interpreted this result to mean that the addition of GTPS drives the protein to a higher state of oligomerization; perhaps from a monomer to a dimer. We show in what follows that this conclusion was qualitatively correct, but that, at the protein concentrations used here (and by Liu and Alberts), the protein exists primarily as a dimer in free solution, and that the addition of PuTP drives the protein either transiently (ATP or GTP) or ``permanently'' (ATPS or GTPS) to a hexameric state. We will show that appreciable concentrations of monomeric gp41 can be observed only at very low protein concentrations (below 1 µM).

Hydrodynamic Measurements

Fig. 2(A and B) shows sedimentation boundaries, obtained in experiments using the Beckman XLA Analytical Ultracentrifuge, for gp41 under standard sedimentation conditions (see ``Materials and Methods'') in the absence and presence of 0.1 mM GTPS, respectively. Under these conditions both boundaries appear quite symmetrical but move down the cell at very different rates, with the sample without nucleotide sedimenting much more slowly than that containing 0.1 mM GTPS. Sedimentation coefficients measured at the midpoints of each boundary are summarized in Table 1, and the distributions of these sedimentation coefficients across each boundary, calculated by the van Holde and Weischet(1978) procedure, are plotted in Fig. 3. Such plots effectively represent the concentration dependence of s(w) from approximately 10% to approximately 90% of the input gp41 concentration (here from 0.5 to 5 µM in gp41 monomers). (^3)(^4)


Figure 2: Traces of sedimentation boundary scans from XLA analytical ultracentrifugation runs. Samples contained 5 µM T4 gp41 protein in the absence (A) and the presence (B) of 0.1 mM GTPS (see ``Materials and Methods'' for details of experimental conditions). Intervals between scans in each experiment were 10 min.






Figure 3: Integral distributions of the sedimentation coefficients of samples along the respective sedimenting boundaries: box, g41p helicase in TAMK buffer; , g41p helicase in the presence of 0.1 mM GTPS in TAMK buffer.



These results show that both forms of gp41 are quite homogeneous at total gp41 (monomer) concentrations down to about 0.5 µM, and that, under these conditions, both samples sediment as fairly monodisperse species. However, as is suggested by the trend toward lower values of s(w) at the lower ends of both sedimentation profiles in Fig. 3, the dominant states of association present under both sets of conditions exist in equilibrium with smaller species, and these smaller species start to make appreciable contributions at the lower concentration ends of the sedimentation profiles. This surmise will be confirmed by experiments described below, in which we show (using protein-protein cross-linking experiments carried out under the same conditions and HPLC experiments at still lower protein concentrations), that gp41 in the absence of PuTPS actually exists as an equilibrium mixture of gp41 monomers and dimers, with dimer representing the most abundant species at protein concentrations >0.5 µM. We also use (see below) protein cross-linking and cryoelectron microscopic methods to show that the larger species (s(w) = 12.2 S) observed in the presence of 0.05-1 mM GTPS corresponds to gp41 hexamers, and suggest that these species are also, at lower protein concentrations, in equilibrium with smaller states of association on the assembly pathway (presumably dimers and tetramers).

In further confirmation of this latter assignment, velocity sedimentation experiments were performed with purified gp41 hexamers that had been cross-linked to completion in the presence of GTPS and then run in the ultracentrifuge in the absence of nucleotides. The sedimentation coefficient obtained for these cross-linked hexamers was very similar to that measured with the non-cross-linked material in the presence of 0.1 mM GTPS (see Table 1); the slightly higher sedimentation coefficient of the cross-linked species probably reflects some molecular compaction resulting from cross-link formation.

Hydrodynamic modeling calculations based on these sedimentation measurements (see below) also strongly support the conclusion that gp41 exists largely as dimers at micromolar protein concentrations, and that these dimers associate to form hexamers in the presence of GTPS. (^5)As we will show below, gp41 dimers can also associate to form hexamers in the presence of the physiological substrates ATP or GTP. However, the gp41 hexamers formed in the presence of these hydrolyzable NTPs are short-lived and redissociate to the largely dimer state after hydrolysis. Thus we conclude that PuTPS can be used to ``freeze'' the otherwise transient PuTP-bound (pre-hydrolysis) state of the helicase. The fact that the non-hydrolyzable substrate analogs, ATPS or GTPS, can drive the hexamerization of this helicase protein also indicates that only binding of ATP or GTP to gp41 is required for hexamer formation, and that hydrolysis of these substrates results in reversion to the largely dimeric state.

HPLC Studies of the Monomer Dimer Equilibrium

As discussed above, velocity sedimentation results suggested that gp41 associates to dimers in solution in the absence of PuTP or nonhydrolyzable PuTP analogs at total protein (monomer) concentrations at the micromolar level or above. Since the limits of detection sensitivity of the UV scanning system prevented the use of lower protein concentrations in the analytical ultracentrifuge, we turned to HPLC molecular exclusion chromatography to characterize the states of association of the gp41 helicase at submicromolar protein concentrations. (^6)A typical set of HPLC experiments, conducted as described under ``Materials and Methods,'' are presented in Fig. 4and show that two HPLC peaks are indeed seen at total gp41 (monomer) concentrations of around 1 µM, with the larger species eluting from the column at 23 min and the smaller at 25 min.


Figure 4: HPLC molecular exclusion chromatograms for gp41 at three selected total protein concentrations. The amounts of gp41 injected into the columns were (respectively): a, 500 pmol; b, 1 nmol; c, 2 nmol. Positions of the dimer and monomer elution peaks are indicated by D and M, respectively. The corresponding elution times are approximately 23 and 25 min for dimer and monomer, respectively. The average total concentration of gp41 in each elution peak are noted.



Variation of the total protein concentration from 100 nM to micromolar concentrations significantly changed the distribution of protein between the two peaks, with the size of the peak corresponding to the larger species increasing with increasing protein concentration, and that of the peak corresponding to the smaller species decreasing in proportion. The larger species became undetectable when the total gp41 concentration in the column fell below 100 nM (data not shown). These results suggest that the larger species (earlier eluting peak) seen in these experiments corresponds to gp41 dimers, and that the smaller species corresponds to gp41 monomers.

The relative changes in the areas of the two peaks with change in total protein concentration also permit us to estimate a monomer dimer association constant. The data from a series of these experiments are plotted in Fig. 5as the fraction of the monomer present versus total protein concentration. When this plot was analyzed as described under ``Materials and Methods,'' a dimerization constant (K) of 0.7 (± 0.2) times 10^6M was obtained. We note that this value should be considered to be approximate, since in addition to the experimental uncertainty contained in the standard error given, conditions within gel filtration columns of this type may not reflect true thermodynamic equilibrium. (^7)However, experiments involving injection of constant amounts of protein at varying protein concentrations and volumes (e.g. 100-µl injection of a 5 µM sample versus 25 µl of a 20 µM sample) showed that the gp41 protein does equilibrate between monomers and dimers, since these procedures produced indistinguishable elution profiles. These HPLC results clearly demonstrate the existence of both gp41 monomers and dimers at these total protein concentrations, and the value of K of 10^6M obtained from the HPLC data is in good agreement with an estimate of this parameter based on protein-protein cross-linking and sedimentation experiments (see below).


Figure 5: Analysis of gp41 dimerization in the absence of nucleotide triphosphate substrates. Data from HPLC molecular exclusion chromatography were plotted using f(m)^2/(1 - f) as a function of the reciprocal of total gp41 protein concentration on elution (see text).



Protein-Protein Cross-linking

Extensive protein cross-linking studies were performed to further characterize the states of association of gp41 in the presence and absence of ATP or GTP (or their nonhydrolyzable thio-analogs). Binding to DNA cofactors (here the DNA replication fork constructs; Fig. 1) greatly stimulates the PuTPase activity of gp41, although a much smaller unstimulated activity can also be measured in the absence of DNA cofactors (Liu and Alberts, 1981a).

Fig. 6A shows that cross-linking to completion at low (200 nM) protein concentrations in the absence of PuTP (or PuTPS) yielded primarily an intramolecularly cross-linked gp41 monomer, which migrated considerably faster in an SDS gel electrophoresis (Fig. 6A) than did the noncross-linked gp41 monomer (presumably due to the molecular compacting and neutralization of positive charges of the protein after cross-linking). In contrast, Fig. 6A also shows that in the presence of GTPS all of the gp41 present associates to form large cross-linked complexes, resulting in a single high molecular weight electrophoresis band. The average size of the complexes present in the high molecular mass band was estimated at >300 kDa, but the exact size (and hence the exact number of gp41 subunits in the complex) could not be determined by this means since the discontinuous SDS-polyacrylamide gel system of Laemmli(1971) used here could not resolve cross-linked gp41 complexes containing more than three subunits (although complexes containing three or less gp41 subunits were advantageously separated by this system).


Figure 6: SDS-polyacrylamide gel electrophoresis to analyze the products of protein-protein cross-linking of g41p by DSP in the absence and presence of nucleotide triphosphates. A, 7.5% (110:1) SDS-PAGE using the gel system of Laemmli (1971). Concentrations of gp41 protein (in total monomers) and DSP in the experiments were 200 nM and 0.15% (w/v), respectively. Times for cross-linking (in min) are indicated at the top of the gel. In the absence of GTPS, the protein monomer was cross-linked intramolecularly and migrates faster than the non-cross-linked protein control (ctrl) lane (see text). B, 3% (30:0.8) SDS-PAGE using the phosphate-SDS gel system of Weber and Osborn(1975) to analyze the gp41 association states in the presence of GTPS. Two sets of reaction conditions were used to permit display of all the reaction intermediates. In lanes 1-3, 5 µM gp41 and 0.01% DSP (from a 0.5% DSP stock solution) were reacted for 1, 5, and 20 min, respectively. In lane4, 5 µM gp41 and 0.1% DSP were reacted for 1 min.



To determine the state of association of the protein under these conditions, and to permit estimation of the concentrations of association intermediates, cross-linking experiments were performed at higher protein and lower cross-linker concentrations so that the number of subunits in the complex could be determined. We used the phosphate-SDS gel electrophoresis system of Weber and Osborn(1975) to separate and visualize the larger cross-linked species. The cross-linked proteins do not migrate exactly according to the logarithm of their molecular weights in this gel, but mixtures of proteins in the size range of interest can be resolved into discrete intermediate bands (e.g. see Finger and Richardson(1982), who used this gel system to study the oligomerization of E. coli Rho protein). Fig. 6B demonstrates the resolving power of this gel system for cross-linked gp41 species and clearly shows the presence of gp41 tetramers, pentamers, and hexamers. Most importantly, and consistent with our quantitative sedimentation velocity results, Fig. 6B shows that the highest association state of gp41 in solution in the presence of GTPS is indeed a gp41 hexamer.

These conclusions were confirmed by the experiments shown in Fig. 7, which were carried out under the same conditions used for sedimentation and show more clearly a ladder of cross-linked intermediates ranging up to hexamers in the presence of GTPS (Fig. 7B), while showing only dimers and monomers in the absence of this nucleotide analog (Fig. 7A). Fig. 7B also shows that no higher species than hexamers are seen even after extensive cross-linking. In addition, the relative intensities of the bands of Fig. 7A show that the system contains mostly dimers and relatively fewer monomers when gp41 was cross-linked to completion under these concentration conditions in the absence of GTPS,, in good agreement with the value of K approx 10^6M estimated for this equilibrium from HPLC data.


Figure 7: Protein-protein cross-linking of gp41 helicase under the same conditions used for the analytical ultracentrifugation experiments: in the absence of GTPS (A) and in the presence of 0.1 mM GTPS (B). Both experiments were carried out by reacting 0.1% DSP for 1, 5, and 20 min., respectively. The reaction products were separated using 3% (30:0.8) phosphate-SDS-polyacrylamide gel electrophoresis.



The hexameric complex observed by cross-linking in the presence of GTPS clearly represents a real structure in solution, since no cross-linked species larger than dimer were seen in control experiments in which cross-linking was carried out with gp41 in the absence of nucleotide (Fig. 7A). This shows that nonspecific (``collisional'') cross-linking does not occur with this reagent at the protein concentrations used in these experiments. On the other hand, since cross-linking to completion preferentially traps the largest associated species present in a dynamic equilibrium, it cannot be determined from these results alone whether the protein exists primarily as hexamers at these protein concentrations or whether (in the presence of GTPS) several different states of association of gp41 coexist in equilibrium. However, the sedimentation results presented above show that gp41, at total protein concentrations of 5 µM, forms a homogeneous (monodisperse) solution of hexamers at GTPS concentrations exceeding 100 µM.

Further examination of the cross-linking patterns of Fig. 7B shows that the amounts of cross-linked dimer and tetramer species significantly exceed those of trimer and pentamer in the late stages of cross-linking. We note that the cross-linking experiments in Fig. 7B were carried out at the same total gp41 and GTPS concentrations used in our analytical ultracentrifugation experiments. As discussed above, we observed a homogeneous 12.2 S complex in analytical ultracentrifugation experiments containing PuTPS, indicating that nearly all the gp41 molecules are present as hexamers. Thus, the cross-linking pattern we observe in Fig. 7B featuring, in particular, the intense dimer band, does not reflect the presence of significant concentrations of this species in the solution. Instead we suspect that this behavior reflects the existence of two different kinds of subunit interfaces within the hexamer structure, resulting in different cross-linking efficiencies at these interfaces. (^8)This cross-linking behavior is very reminiscent of the distribution of cross-linked species obtained in similar experiments with the E. coli transcription termination factor Rho, which also exists in its functional form as a hexameric helicase formed by the association of three asymmetric dimers (Seifried et al., 1991; Geiselmann et al., 1992a, 1992b). As with Rho, this finding suggests that the assembly of gp41 subunits as a consequence of PuTP binding probably follows primarily a monomer dimer tetramer hexamer pathway. (See ``Discussion'' for further comparative consideration of these helicases.)

Finally, we have used cross-linking experiments to look for any additional effects on the association states of gp41 that might result from the addition of adding single-stranded or forked DNA templates, or nucleotide diphosphates (the product of gp41 ATPase/GTPase activity). We find (data not shown) that the presence of these molecules has little effect on the oligomerization equilibria of gp41 assembly.

Structure of the Hexameric gp41 Helicase

Hydrodynamic Modeling

The relationship between the frictional coefficient (or sedimentation coefficient) that characterizes a particular macromolecule and the same parameter for higher states of association of the same macromolecule can be used to determine some aspects of the structure of the associated complexes (Kirkwood, 1954; van Holde, 1975). When this Kirkwood modeling approach is applied to the analysis of the structure of a hexamer complex formed from identical subunits (assuming no major changes are induced in the frictional characteristics of the subunits as a consequence of the assembly process), only a limited number of possible structures need to be considered. This follows because the possibilities are limited by the structural symmetry requirements involved in assembling hexamers from asymmetric dimer units. The hexameric structures that are possible are pictured in Fig. 8. We note that the linear hexamer shown as Structure A, while not symmetrical, is included here for completeness since it represents the simplest possible hexamer. Additionally, Structure D is taken to represent the many possible structures of this symmetry, which differ in thickness and overall ring size. The other structures are discrete. We note that Structures C and E represent the extremes of structures of type D.


Figure 8: Models and results of hydrodynamic modeling of the gp41 dimer and the various forms of gp41 hexamers, using the methods of Kirkwood(1954) and van Holde(1975). Calculated sedmentation coefficients for the various forms of gp41 complexes are indicated below their respective proposed conformations.



Our sedimentation experiments yielded a sedimentation coefficient (s(w)) of 5.7 S for gp41 in the absence of GTPS. This value of s(w) is very close to the theoretical value for a dimer of this size (5.76 S), which is consistent with our finding that gp41 forms dimers with an association constant of about 10^6M. This follows because our analytical ultracentrifugation experiments were carried out at total gp41 concentrations of 5 µM. Therefore the protein should be mostly in the dimer form, in equilibrium with only minor amounts of monomer. This conclusion is also supported by the observed integral distribution of sedimentation coefficients, which slopes downward to about 4.5 S at protein concentrations below 1 µM (see Fig. 3).

The experimental sedimentation coefficient of 12.2 S for gp41 in the presence of GTPS is best fit by a structure of type D shown in Fig. 8, although (since the subunits of real macromolecules would be expected to interact over a surface rather than merely at single points) a slightly compressed version of Structure C would also fit this experimental parameter. Thus our sedimentation results and calculations suggest that the arrangement of gp41 subunits in the hexamer structure is likely to resemble C, with all six subunits lying on a ring within one plane, or D, with all six subunits lying around a ring with three subunits in an upper plane and three in a lower plane. As will be shown below, the structure of hexameric gp41 as determined by cryoelectron microscopy is in total accord with this conclusion.

Cryoelectron Microscopy

We have used cryoelectron microscopy to visualize directly the shape of the gp41 hexamer. This procedure is particularly advantageous for labile complexes since the ultrafast cooling process employed in this method freezes the complexes ``instantaneously'' without requiring chemical fixatives, shadowing, or the other potentially perturbing aspects of conventional electron microscopy. A weakness of the cryoEM technique, which applies equally to any transmission electron microscopic method, is that certain populations of complexes may preferentially bind to the carbon support surface, while others may localize primarily within the holes in the carbon where the images are examined. However, the hydrodynamic studies described above show that populations of gp41 complexes in solution under the conditions of this study are monodisperse, indicating that this reservation need not concern us here.

In the absence of purine nucleotide triphosphates or their analogs, images of gp41 revealed no reproducible structures other than small dots, which sometimes appeared as arcs (see arrows in Fig. 9A). These particles have an apparent diameter of approximately 20 A, and although they could not be measured reliably under the imaging conditions that we have used because structural information at this spatial resolution is convoluted by the contrast transfer function of the electron microscope (Erickson and Klug, 1971), our results from sedimentation and HPLC studies (above) suggest that these particles probably correspond to gp41 monomers and dimers. Occasionally somewhat larger particles (50-60 A in diameter) were observed, which might correspond to disordered aggregates of the dots shown in Fig. 9A.


Figure 9: Cryoelectron micrographs of g41p helicase complexes formed: in the absence of nucleotide triphosphate (A), in the presence of ATP (B), and in the presence of both ATP and a DNA replication fork construct (C). The same magnification factors apply to all panels of this figure; the bar in panelA corresponds to 1000 A.



Addition of PuTP or PuTPS to gp41-containing solutions resulted in the appearance of the larger and much more easily visible particles corresponding to the circularrings shown in Fig. 9B. These toroidal structures are approximately 100 A in diameter; much larger than any of the smaller particles seen in the absence of nucleotides. (Some of the latter type of particles are still occasionally visible in the presence of PuTPS, but at much reduced numbers.) The rings formed in the presence of ATP (or GTP) and ATPS (or GTPS) appear identical, and no such structures are seen in the presence of ADP alone.

The structures formed with the nonhydrolyzable nucleotide triphosphate analogs seem to be indefinitely stable (i.e. at least for 20 min). However comparable structures induced by the addition of ATP had a much shorter lifetime, perhaps reflecting the depletion by gp41-catalyzed hydrolysis of the available PuTPs. To test this point solutions containing 5 µM gp41 were incubated with an initial concentration of 1 mM ATP, and specimens were prepared after 1-2 min, and again after 15-18 min. While numerous rings were present at the earlier time, none were visible at the 15-18 min, when nearly all of the available ATP had been hydrolyzed. The addition of another aliquot of 1 mM ATP to this same sample caused the reappearance of the ring structures (after an additional 1-2 min of incubation).

Addition of the miniature DNA forks to gp41 in the absence of nucleotide triphosphates (or their PuTPS analogs) did not cause the appearance of the rings or any other structures, and the images obtained are indistinguishable from the small particles (dots) seen with gp41 alone. In the presence of both the miniature DNA forks and nucleotide triphosphates, rings similar to those seen with nucleosides in the absence of DNA cofactors were abundant (Fig. 9C). In the presence of DNA, however, the majority of the rings were very clearly hexagonal, with pronounced vertices. An occasional hexagonal ring of this type was sometimes present in the populations of rings obtained with gp41 containing nucleotides in the absence of DNA (Fig. 9B), but these were infrequent, whereas they seem to make up the majority of particles in the specimens obtained in the presence of the DNA forks and ATP. Substitution of the nonhydrolyzable thio-analogs for NTP yielded images indistinguishable from those obtained in the presence of the NTP substrates. Preliminary observations suggest that the observed change in the appearance of gp41 hexamer (from roughly circular to distinctly hexagonal) can also be brought about by single-stranded DNA oligonucleotides in the presence of NTP, and thus may not require the presence of a model DNA replication fork construct.

Computer-based analysis of images (Fig. 10) of the ATP-induced gp41 structures, in the presence and absence of DNA forks, confirms these direct visual observations. In both cases six protein subunits can be clearly seen in the ring-shaped complexes. Comparison between these images also provides additional structural details and reveal subtle differences between the two complexes. The structure of the gp41 hexamer in the presence of the miniature DNA forks is clearly hexagonal, with all the subunits clearly separated (Fig. 10B), while the hexamer visualized in the absence of the DNA fork appears more ``doughnut-like'' (Fig. 10A). Also, the structure formed by gp41 in the presence of the DNA fork appears slightly larger in diameter (20%), suggesting that the protein subunits are ``stretched out'' from the center of the structure by DNA binding (Fig. 10B). In addition, the center of the complexes seems to be less open (denser) when the DNA fork is bound (Fig. 10, compare A and B).


Figure 10: Averaged image of the gp41 helicase hexamers in the presence of ATP (A) and both ATP and a miniature DNA replication fork (B). The images from the original cryoelectron micrographs were first digitized and then aligned (both translationally and rotationally) and computer-averaged (see text). Over 200 individual images were averaged in each case.




DISCUSSION

Assembly Pathway of the gp41 Helicase

In this study we have described the assembly of the PuTP-activated gp41 helicase from its constituent subunits, and have shown that this activated species is a hexamer of identical subunits in a toroidal arrangement. A schematic summary of the assembly process is shown in Fig. 11. We have also shown that the gp41 helicase of bacteriophage T4 exists largely as a dimer of gp41 subunits in the absence of PuTP cofactors under physiological solution conditions and gp41 concentrations, and that this dimer is in labile equilibrium with its monomeric constituents. The monomer dimer equilibrium is characterized by an association constant of 10^6M, and the existence of the gp41 dimer in the absence of significant concentrations of higher forms shows that dimer formation involves an asymmetric ``head-to-head'' interface between monomer subunits, and that these head-to-head interfaces involve tighter binding interactions than those between the putative ``tail-to-tail'' interfaces that must form if oligomerization is to proceed beyond the dimer level. The absence of measurable quantities of higher species in either the sedimentation or the cross-linking experiments suggests that the putative tail-to-tail association constant for the assembly of dimers to tetramers and higher species (K) must be less than 10^4M in the absence of PuTP cofactors.


Figure 11: A schematic diagram of the association pathways used in the assembly of the gp41 helicase in the absence and in the presence of nucleotide triphosphate substrates.



However, on binding of ATP or GTP, these dimers are driven to associate to a hexameric state, which redissociates to dimers after depletion of the PuTP by the intrinsic PuTPase activity of the gp41 helicase. The hexameric state can be stabilized and made ``permanent'' (and thus more easily amenable to structural study) by binding the essentially nonhydrolyzable PuTP analogs (and PuTPase inhibitors) ATPS and GTPS to the nucleotide binding sites of the gp41 molecules. Since PuTP hydrolysis, single-stranded DNA translocation, and double-stranded DNA unwinding are concurrent activities of the gp41 helicase, the gp41 hexamer formed upon binding to ATP or GTP is likely to represent the active form of this helicase.

Protein-protein cross-linking studies and cryoelectron microscopy have suggested that the assembly process of the ATP/GTP induced hexamer follows a dimer tetramer (open hexamer) closed hexamer pathway, as outlined in Fig. 11, while hydrodynamic modeling calculations and cryoelectron microscopic observation suggest that the end product of this pathway is a hexagonal structure of type C or D, shown schematically in Fig. 8. The relationship between the abundance of the gp41 hexamer species and total concentration of gp41 subunits also suggests that PuTP (or PuTPS) binding strengthens both the head-to-head and the tail-to-tail interactions within and between gp41 dimers. This change must be particularly large for the interaction across the putative tail-to-tail interface that assembles the gp41 dimers to hexamers, since the association constant for this interface (K) must increase from <10^4M to >10^7M in order to drive hexamer formation to completion at the experimental concentrations of gp41 protein used. This means that PuTP binding must increase the free energy of association between gp41 dimers from less than -6 kcal/mol to more than -9 kcal/mol. The molecular basis of how this change in binding free energy is achieved at the tail-to-tail interface has not been defined.

Cross-linking studies have also shown that the ``active'' gp41 hexamer is neither further stabilized or destabilized by the binding of single-stranded DNA (or DNA ``fork'' constructs), although cryoEM observations suggest that the molecular structure of the hexamer may be altered somewhat by interaction with such DNA substrates (the hexamer is driven from a more doughnut-like to a more sharply hexagonal state on binding DNA; see ``Results''). Since DNA binding does greatly stimulate the intrinsic PuTPase activity of gp41, this functional alteration may be reflected in these minor structural changes we have observed with cryoEM.

Functional Implications of the Hexagonal Structure of the Active gp41 Helicase

The results of these structural studies of the gp41 replication helicase of T4 are very reminiscent of comparable studies performed previously in our laboratory on the E. coli transcription termination factor Rho (see Geiselmann et al.(1993), for a summary of earlier work and a proposed functional model), which is thought to function in Rho-dependent termination as an 5` 3` RNA-DNA helicase. The active form of Rho is also a ring-shaped hexamer of identical subunits, that shows a virtually identical cryoEM structure to that adduced here for the hexameric form of gp41 (Gogol et al., 1991). (^9)In addition the assembly pathway from dimers appears to be much the same (Seifried et al., 1991), although for Rho the inter-dimer affinity is strong enough to drive hexamer formation virtually to completion in the absence of ATP or nucleic acid cofactors. Studies of Rho also have shown that the functional entity is an asymmetric dimer (Seifried et al., 1992), and that the Rho hexamer displays D3 symmetry. In addition the active Rho hexamer carries three strong and three weak ATP and RNA binding sites (Geiselmann and von Hippel, 1992; Geiselmann et al., 1992c; Wang and von Hippel, 1993), which may interconvert on ATP hydrolysis, leading to partial release of the bound RNA and to directional translocation of the Rho hexamer along the RNA (Geiselmann et al., 1993). The existence of such asymmetric binding affinities for the putative ATP and DNA binding sites of the gp41 hexamer has not yet been investigated.

However, it is tempting to speculate that the similar structures of the active Rho and gp41 hexamers may reflect functional similarities as well. Rho is thought to translocate unidirectionally (5` 3`) along single-stranded RNA (Geiselmann et al., 1993; Steinmetz and Platt, 1994) and steady-state kinetic analysis of its ATPase activity is consistent with the notion that gp41 does translocate unidirectionally (5` 3`) along single-stranded DNA (Young et al., 1994c). The details of the more complex interactions of both of these proteins when working within the functional transcription and replication complexes remain to be elucidated, but the hexameric structure of these helicases may have general mechanistic significance.

This speculation is strengthened by the collection, in Table 2, of the properties of a variety of prokaryotic and eukaryotic helicases that have subjected to some structural investigation in recent years. This table represents an updating of two shorter similar sets of data that were presented earlier by Matson and Kaiser-Rogers(1990) and by Lohman(1993). As Table 2shows, these various helicases function within the contexts of complex DNA replication, transcription, recombination, and repair processes, and perhaps of RNA splicing processes as well, and the details of how they operate may differ quite appreciably from one to another. However the prevalence of hexamers among these helicases is striking, and in the limited number of cases where actual structural studies have been performed, the ring-shaped hexamer motif seen with gp41 and Rho also seems to be present (see the studies of T7 gp4 by Hingorani and Patel (1994), E. coli RuvB by Stasiak et al.(1994), and E. coli DnaB by Bujalowski and Klonowska(1993, 1994). It will be interesting to see whether common functional mechanisms emerge as molecular studies of these helicases, operating both alone and as parts of biologically relevant complexes, are carried further. ()



Functional Integration of gp41 Helicase into the Primosome and the T4 DNA Replication Elongation Complex

Relatively little can currently be said about the mechanistic details of the functional integration of the gp41 helicase with its primosome partner, gp61, and the replication complex as a whole. It is known that gp41 is active as a multimeric complex that interacts with a monomer of the gp61 protein (Hinton and Nossal, 1987; Richardson and Nossal, 1989a). We now demonstrate that the PuTP-activated complex of gp41 is a ring-shaped hexamer. This activated form of gp41 is capable of forming a stable complex with gp61 on appropriate DNA templates with 6:1 subunit stoichiometry, ()and we assume that these interactions must somehow be congruent with the hexagonal organization of the gp41 helicase alone. Recent studies (Spacciapoli and Nossal, 1994; Barry and Alberts, 1994; Morrical et al., 1994) suggest that the T4-coded gp59 protein may also interact functionally with gp41. This protein, under certain conditions, acts as a gp41 helicase assembly factor and is essential for the loading of the gp41 helicase onto gp32-covered single-stranded DNA to form a functional complex with other replication proteins. Clearly much of what gp41 and its (often hexagonal) analogs do biologically involves ATPase-driven translocation along single-stranded nucleic acid substrates, but the details of the processes that these proteins engage in, and of how their putative helicase functions are integrated into the overall biological processes in which they play such important roles, remain, in almost all cases, to be elucidated.


FOOTNOTES

*
These studies were supported in part by United States Public Health Service Research Grants GM-15792 and GM-29158 (to P. H. v. H.), by Damon Runyon-Walter Winchell Cancer Research Fellowship DRG-1152 (to F. D.), by a grant to the Institute of Molecular Biology at the University of Oregon from the Lucille P. Markey Charitable Trust, and by National Science Foundation Grant MCB-9303880 (to E. P. G.). P. H. v. H. is an American Cancer Society Research Professor of Chemistry. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: gp(number), gene product (number); cryoEM, cryoelectron microscopy; DSP, dithiobis(succinimidyl propionate); DTT, dithiothreitol; , molar extinction coefficients at (nm); HPLC, high performance liquid-phase chromatography; PuTP, purine nucleotide triphosphate (i.e. ATP or GTP); PuTPase, purine nucleotide triphosphatase, ATPase or GTPase; PuTPS, ATPS or GTPS; GTPS, guanosine 5`-3-O-(thio)triphosphate; TEMED, tetramethylethylenediamine.

(^2)
The purification procedure involves successive ammonium sulfate precipitations of the protein; we found that the protein could not always be completely resolubilized after these precipitations. In addition the original protocol recommended storing the protein as frozen aqueous stock solutions at -80 °C; we found that freeze-thaw cycling resulted in inconsistencies in protein concentration and activity. These problems were alleviated by using a HEPES-based buffer in place of Tris-based buffer and acetate salts in place of chloride salts in the purification procedures, and by storing the purified protein unfrozen at -20 °C in a buffer containing 50% (v/v) glycerol.

(^3)
We have shown that ATPS and GTPS are equally effective in shifting this equilibrium. Young et al. (1994c) showed that ATP and GTP are also approximately equally active as substrates for the DNA-stimulated PuTPase of gp41 (i.e. they exhibit the same K and k); Liu and Alberts (1981a) had earlier observed that GTP has 2-3-fold more PuTPase activity under some conditions.

(^4)
Plots such as those of Fig. 3lose accuracy at the extremes of the fraction boundary, since sedimentation boundary scans contain larger errors near the plateaus and base lines. On the other hand, data points in the middle of the boundaries (usually between 5 and 95% or 10 and 90%, depending on the level of noise in the scans) have very small standard errors and accurately reflect the sedimentation behavior of the complexes.

(^5)
We also attempted to confirm these conclusions by performing sedimentation equilibrium measurements, but gp41 helicase proved to be unstable at room temperature over the long times required to reach sedimentation equilibrium for proteins of this size.

(^6)
Seifried et al.(1991) used a similar technique to analyze the states of association of Rho, the transcription termination RNA-DNA helicase of E. coli.

(^7)
We note that the monomer dimer equilibrium must be partially ``frozen'' within the column itself, since otherwise two separate peaks would not have been expected on the separation time scale of the HPLC experiments, which is quite comparable to the time scale of the sedimentation experiments.

(^8)
Of course this conclusion is also consistent with our finding that gp41 subunits associate only to the level of dimers in the absence of PuTP or its nonhydrolyzable analogs. If there were only one type of subunit interface present under these conditions significant concentrations of higher order species would have been revealed, both in these cross-linking experiments and in the sedimentation velocity experiments.

(^9)
The hexameric complexes visualized for the E. coli transcription termination factor Rho and T4 gp41 helicase by cryoEM are very similar in subunit arrangement and size. The only difference we have seen is that in the presence of certain RNA oligomer substrates Rho can form dodecamers (a double hexamer structure with one haxamer ring on top of the other), while such structures have not been observed with gp41 in the presence of either the DNA replication fork constructs or single-stranded DNA.

()
We note that a similar ligand-induced assembly of subunits has been seen with the Rep helicase of E. coli, for which DNA binding seems to drive association from the monomer to the dimer state (Chao and Lohman, 1991). In addition we note that these workers (Chao and Lohman, 1991; T. Lohman, personal communication) have examined the state of association of the Rep helicase under a wide variety of cofactor and solution conditions and have not observed hexamer formation. As a consequence they have concluded that the Rep helicase functions as a dimer and have put forward a helicase model based on a dimer mechanism (Lohman, 1992, 1993).

()
F. Dong and P. H. von Hippel, manuscript in preparation.


ACKNOWLEDGEMENTS

We are grateful to Drs. Nancy Nossal and Bruce Alberts and their laboratories for cells and plasmids and for helpful discussions, to Drs. Michael Reddy and Mark Young for discussions and experimental help, to Steve Weitzel for experimental assistance, to Deborah McMillan and the University of Oregon Biotechnology Laboratory for help with the HPLC experiments, to Drs. Kensal van Holde and Karen Miller of Oregon State University for access to the Beckman XLA Analytical Ultracentrifuge, and to Wen Xiong of University of Texas at Dallas for assistance in the initial computer imaging work.


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