Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex

J. Mol. Biol. (1992) 224, 395-412

Cryoelectron Microscopic Visualization of Functional Subassemblies of the Bacteriophage T4 DNA Replication Complex Edward P. Gogol?, Mark C. Young, William L. Kubasekl Thale C. Jarvisg and Peter H. von Hippel Institute oj Molecular Biology and Department of Chemistry University of Oregon, Eugene, OR 97403, U.X.A. (Received 3 August

1991; accepted 26’ November 1991)

A specific complex of proteins involved in bacteriophage T4 replication has been visualized by cryoelectron microscopy as distinctive structures in association with DNA. Formation of these structures, which we term “hash-marks” for their characteristic appearance in association with DNA, requires the presence of the T4 polymerase accessory proteins (the products of T4 genes 44, 45 and 68), ATP and appropriate DNA cofactors. ATP hydrolysis by the DNA-stimulated ATPase activity of the accessory proteins is required for visualization of the hash-mark structures. If ATP hydrolysis is stopped by chelation of Mg2+, by dilution with a non-hydrolyzable ATP analogue, or by exhaustion of the ATP supply, the DNA-associated structures disappear within seconds to minutes, indicating that they have a finite and relatively short lifetime. The labile nature of the structures makes their study by more conventional methods of electron microscopy, as well as by most other structural approaches, difficult if not impossible. Addition of T4 gene 32 protein increases the number of hash-mark structures, as well as increasing the rate of ATP hydrolysis. Using plasmid DNA in either a native (supercoiled) or enzymatically modified state, we have shown that nicked or gapped DNA is required as a cofactor for hash-mark formation. Stimulation of the ATPase activity of the accessory proteins has a similar cofactor requirement. These conditions for the formation and visualization of the structures parallel those required for the action of these complexes in promoting the enzymatic activity of the T4 DNA polymerase, as well as the transcription of late T4 genes. Substructure in the hash-marks has been examined by image analysis, which reveals a variation in the projected density of the subunits comprising the structures. The threedimensional size of the h.ash-marks, modeled as a solid ellipsoid, is consistent with that of the gene 44162 protein subcomplex. Density variations suggest an arrangement of subunits, either tetragonal or trigonal, viewed from a variety of angles about the DNA axis. The hashmark structures often appear in clusters, even in DNA that has a single nick. We interpret this distribution as the result of one-dimensional translocation of the hash-marks along the DNA after their ATP-dependent initial association with, and injection into, the DNA at nicks or gaps.

Keywords: T4; replication;

protein-DNA complexes; image analysis

cryoelectron

microscopy;

1. Introduction t Present address: Program in Molecular and Cell Biology, University of Texas at Dallas, Mail Station

Some years ago, Alberts and Nossal and their co-workers showed that a complex of seven bacteriophage T4 proteins can be reconstituted in k?ro, and that this complex is able to carry out the elongation phase of leading and lagging strand IDNA replication (Barry & Alberts, 1972; Morris et al., 1979a,b; Nossal, 1979; Nossal & Alberts, 1983). The measured rate of synthesis and the fidelity and

F03.1, Richardson, TX 75083, U.S.A. $ Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, U.S.A. 3 Present address: Department of Cellular, Developmental and Molecular Biology, University of Colorado, Boulder, CO 80309, U.S.A.

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processivity of this reaction were comparable to those manifested by the T4 DNA replication system system in wivo (Liu et al., 1978). This seven-protein includes the T4 DNA polymerase (g43pf), the T4 single-stranded DNA binding (g32p) protein, the T4 polymerase accessory (g44/62p and g45p) proteins, and the T4 helicase and primase (g41p and g61p, respectively). It was also shown by the same workers that a five-protein subset of this replication complex (lacking the helicase and primase) can perform continuous strand synthesis on a primed single-stranded DNA template, again with physiological rate, fidelity and processivity. The mechanism by which this complex carries out DNA replication has been studied extensively as a model system for replication, since activities of functional subcomplexes from the T4 system have counterparts in more complex organisms (Crute et d., 1990; Downey et al., 1990; McHenry, 1988). Our laboratory, along with others, has since been involved in attempting to obtain a more detailed understanding of the macromolecular interactions that underlie the structure and function of these complexes in T4 DNA replication. Structural and enzymatic study of the polymerase accessory proteins complex (Jarvis et al., 1989a,b, 1990, 1991) has shown that subassemblies of this complex are found in solution, uncomplexed to DNA. A tight g44/62p complex has been identified, which consists of four as well as a g45p g44p and one g62p subunits, assembly that is composed of a trimer of g45p subunits (Jarvis et al., 1989a). The combination of these two protein complexes has a DNA-dependent ATPase activity that is essential to its biological function and binding specificity (Piperno & Alberts, 1978). Associated with DNA, the complex of accessory proteins, presumably formed by a combination of the two subassemblies, comprises a “processivity assembly” or “sliding clamp” that interacts with the T4 DNA polymerase, in the presence of g32p and ATP, to form a holoenzyme complex (Newport et al., 1980; Huang et al., 1981; Munn, 1986; Jarvis et al., 1991). This complex can perform DNA synthesis under physiological salt conditions with a processivity that is greatly enhanced over that of the polymerase alone (Jarvis et al., 1991). A second role for the replication accessory proteins has been identified more recently. Transcription of certain T4 genes occurs only during late stages of the infection cycle, and is coupled to replication of the T4 genome (Geiduschek et al., 1983). The transcription of these T4 late genes requires the T4-modified Escherichia coli RNA polymerase and several T4 gene products, among them g45p. All three of the T4 polymerase accessory proteins (g45p and g44/62p), along with ATP hydrolysis, are necessary for in vitro stimulation of trant Abbreviations used: g43p, g44/62p, g45p and g32p, products of the T4 genes 43, 44 and 62, 45 and 32; g2p, product of phage fl gene II; RF-Ml3 DKA, replicativeform (double-stranded) Ml3 DNA; BME, fi-mercaptoethanol.

scription of late T4 genes (Herendeen et a$., 4989). This transcription stimulation also requires a nick in the double-stranded DNA, a site that is a probable initial binding or assembly site for the complex of accessory proteins. This complex appears to serve as a stimulatory factor specific for late T4 promoters, as the affected genes can be hundreds of base-pairs away in either direction from the initial binding site at the nick. Enhancement of transcription from a variant T4 late promoter requires t,hat the nick be on the non-transcribed strand, suggesting a differential association with one half of the double helix. The proteins interact, wit,h the RNA polymerase, an interaction mediated by another T4-encoded protein, g33p (Herendeen et al., 1990). The replication accessory proteins thus are a vital link in replication-transcription coupling in T4 phage synthesis. Characterization of the physical nature of these proteins, and of their interactions with DNA, may prove critical in understanding the mechanism of mobile transcription enhancers as described by Herendeen et al. (1989: 1990). Structural characterization of the complex formed by the replication proteins with DNA has been difficult because of their transient and chemically sensitive nature. We have attempted to visualize DNA-bound assemblies of the replication accessory proteins directly using condit,ions under which they exhibit full activity, by cryoelectron microscopy of amorphously frozen specimens, This technique permits the examination of functional macromolecular complexes at moderate resolution without the addition of potentially disruptive agents (Dubochet et al., 1988), and has been used to study both individual proteins (Gogol et al., 1989a,6) and protein-RNA complexes (Gogol et al., 1991), as well as to visualize naked DNA directly (Adrian et al., 1991 f Furt’hermore, the very rapid freezing rates used in specimen preparation cause the trapping of transient and potentially unstable states of macromolecular assemb!ies, which are difficult or impossible to prepare for examination by other structural methods. Analysis of individual images, visual inspection or computerized by either methods, permits the identification of these states, as well as intermediates in which macromolecular rearrangements have occurred (e.g. see Gogol et a.l., 1990). In an effort to learn more about the “proeessivity complex” formed by the T4 accessory proteins, we have used this structural approach to visualize the ATP-induced assembly of the accessory proteins wit’h DNA. We have explored the effect of changing the physical characteristics of the DNA cofactor in these assemblies, and have used image analysis to characterize the distinctive complexes.

2. Materials (a) Proteins

and Methods and DNA

T4 replication proteins were isolated both from T4-infected E. coli cultures as previously described (Morris et aZ., 1979a; Ja,rvis et al., 1989a), or from the induced overexpression of the cloned genes. In the lat,ter

T4 Accessory Protein-DNA case, g45p and g44/62p were purified from E. coli strains HBlOl (F-, hsdS20 (r;, m,), ara-14, proA2, lacY1, GalK2, rpsL20 (SG), ~~1-5, mtl-1, supE44, recA13, A-) (Messing, 1983), containing the plasmid pTL151W (Rush et al., 1989). This strain and plasmid were generous gifts from Drs T.-C. Lin and W. H. Konigsberg, Yale University. The proteins were isolated by a procedure to be described (M. Young and M. Reddy, unpublished results). The proteins were stored in 50% glycerol at either - 20°C (up to 6 months) or - 70 “C (longer storage). Protein concentrations were determined from absorbance measurements at 280 nm, using the absorption coefficients previously calculated for each protein (Jarvis et al., 1989). The phage fl initiator protein, the product of fl gene II (g2p), was isolated from E. co& strain K561 (Lyons & Zinder, 1972) containing the plasmid pDGl17IIA (Greenstein & Horiuchi, 1987), which includes the fl gene II under transcriptional control of the ZacI promoter. The strain was a generous gift from Dr David Greenstein, Rockefeller University. g2p was isolated by the method of Greenstein & Horiuchi (1987), omitting the final gel filtration step. The protein was judged to be more than 90% pure by SDS/polyacrylamide gel electrophoresis and staining with Coomassie blue, and was contaminated only by a trace of the fl gene X protein. After purification the protein was stored at concentrations of approximately 907 to @I3 mg/ml in 12 mlcl-imidazole, 200 mivr-KCl, 65 mM-EDTA, 2 rnM-2-mercaptoethanol @ME), 50% (v/v) glycerol (pH 6.8) at -70°C. The g2p concentration was estimated by absorbance at 280 nm, using an s2s,, of 615 ml mg-i cm-l, calculated from the amino acid composition of g2p and the amino acid extinction coefficient of Edelhoch (1967). Replicative form (RF) bacteriophage Ml3 DNA was prepared by M13mp19 infection of E. coli strain JMlOl (Stratagene) using standard protocols (Maniatis et al., 1982). DNA was isolated by the alkali lysis procedure, followed by extraction with chloroform/phenol (50%, v/v) and precipitation with ethanol (Maniatis et al., 1982). The RF-Ml3 DNA was stored in TE buffer (10 mw-Tris . HCl (pH 7.0), 1 mM-EDTA) for approximately 2 to 4 years at 4°C prior to use in the experiments reported here. Agarose gel electrophoresis revealed the DNA to be a mixture of supercoiled and relaxed forms. Plasmid DNA was prepared from E. coli cultures transformed with the 2958 base-pair cloning vector BlueScript SK+ (Stratagene), which contains the fl origin of replication. Various recombination-defective E. coli strains were used to grow the BlueScript in an attempt to minimize the oligomerization of the plasmid, including the JM109 and SURE strains (Stratagene) and LU-1 (gift from Eric Foss, University of Oregon). Cultures (1 to 4 1) of E. coli were grown overnight and DNA was prepared by the cesium including alkali/SDS extraction procedure, chloride density-gradient centrifugation (Maniatis et al., 1982). The plasmid was dialyzed into TE buffer and stored at 4°C. Agarose gel electrophoresis revealed the DNA to be a mixture of monomeric and multimeric (primarily dimer, with smaller amounts of trimer and tetramer present) forms of supercoiled plasmid, with trace amounts of relaxed or linear plasmid only occasionally present. Digestion with EcoRI (Bethesda Research Labs (BRL)) yielded a single band of approximately 3000 base-pairs. (b) Nicked and gapped plasmid DNA Singly nicked plasmid was produced by treating the supercoiled BlueScript with fl g2p, which nicks super-

397

Complexes

coiled DNA at the fl origin of replication (position 163 in BlueScript SK + ). Samples of the g2p were thawed and diluted to twice their volume with water, concentrated buffer and DNA to produce a final concentration of 50 miw-Tris. HCl, 100 mrvr-KCl, 6 mi?-imidazole, 3 mM5 mm-dithiothreitol (DTT), 025 mM-EDTA, MgCL, 1 mM-/?ME, 25% (v/v) glycerol, pH 8.5 (including the g2p storage buffer components) and 0.1 to 0.25 /*g/ml DNA. The ratio of DNA to g2p used in these preparations varied from 1.4 to 30 pg DNA/pg g2p. The reactions were incubated at 37°C for 2 to 35 h and stopped by the addition of excess EDTA. The DNA was extracted with phenol/ chloroform (50%, v/v) precipitated with ethanol, and dissolved in TE buffer. Gaps were introduced in the nicked plasmid DNA in a 3’ to 5’ direction from the nick site by digestion with exonuclease III. Nicked plasmid was diluted with buffer (50 mr\l-Tris*HCl (pH 8.0), 10 mM$ME, 5 m&r-MgCl,,) to a concentration of 1 mg/ml, and exonuclease III (BRL or New England Biolabs) was added at a ratio of 1 unit/2 pg DNA. The specimens were incubated at 37°C for various times (usually 5 to 15 min). Reactions were stopped by addition of EDTA to 10 mM, and the DNA was extracted with phenol/chloroform and precipitated with ethanol. The extent of digestion by exonuclease III was examined by determining the time (approximately 30 min under the above conditions) required to digest the 594 bases in t.he 5’ direction from the nick site to the RsaI site at posiltion 2526 bases in BlueScript. Digestions for 5 min are therefore estimated to have removed - 100 bases; producing a single-stranded gap of approximately that size in the plasmid. Linear fragments of plasmid DNA, either intact, nicked or gapped, were made by digestion with the restriction endonuclease RsaI (International Biotechnologies, Inc.), which cuts BlueScript at positions 655 and 2526. ‘This digestion yields 2 fragments, one of 1008 base-pairs containing the fl origin (nick/gap site) approximately at its center, and a second of 1870 base-pairs lacking the fl origin. Plasmid (1 to 2 mg/ml) was mixed with Rsal (10 units/mg DNA) in the supplied buffer and incubated for 1 h at 37 “C. Reactions were stopped by addition of EDTA (20 mM or more), and the DNA was extracted, preeipitated and dissolved in TE buffer as described above. Agarose gel analysis revealed that only 2 fragments were produced, approximately 1000 and 1800 base-pairs in length. (c) GeZ electrophoresis

DNA preparations were routinely analyzed by electrophoresis in 1.0 to 1.5% agarose gels, run in 40 mlur-Trisacetate (pH 7.5), 1 mM-EDTA. Digestion products of the g2p-treated DNA were denatured in 95% (v/v) formamide, 20 mM-EDTA and analyzed by electrophoresis in a 44% acrylamide, 8 M-urea gel, run in 90 mrvr-Tris-borate (pH 7.5), 2 mM-EDTA at 45°C for -6 h. Gels were soaked in a 1 pg ethidium bromide/ml solution and bands were visualized by fluorescence under ultraviolet light. (d) ATPase

assays

The ATPase activity of the g44/62p was measured in the mixture of the 3 accessory proteins, in both the presence and the absence of g32p by the spectrophotometric assay described by Panuska & Goldthwait (1!380). The proteins were diluted into 1 ml cuvettes containing 25 m&r-Hepes (pH 7.5), 160 mM-potassium acetate, 6 m&I1 mM-phospho-(enol)magnesium acetate, 5 mM-DTT,

E. P. Gogo et a?

398

pyruvat,e, 0% ma-NADH? 1 rn~-ATP, and 8 units each of pyruvate kinase and lactate dehydrogenase (Sigma), pre-equilibrated to 37 “C. The final protein concentrations used were: 0.1 PM-g44/62p complexes, 1 pM-g45p trimers and 1 ptil-g32p. Absorbance changes at 340 nm were recorded to determine the ATPase activity in the absence of DNA. DNA samples were then added t’o a final concentration of 10 to BOpg/ml, and absorbance changes were once again monitored. Linear regions of the time course of absorbance readings were used to determine both the DNA-independent and DNA-stimulated ATPase rates for each assay. In some cases, rates were determined from absorbance changes using a A&340nm of 6300 M-l cm-’ (Buetler & Supp, 1983).

(e) Prepuration

of sa;mples jor electron microscopy

Proteins were thawed immediately before dilution with KTMB buffer (60 rnx-potassium acetate; 25 mM-Trisacetate, 6 rnM-magnesium acetate, 5 mM#ME, pH 75) of DNA. Typical concentrations used were: and addition @15 PM-g44/62p complex. 1.5 pM-g45p trimers, 3.7 +LUWg32p (monomers), 0.2 mg DNA/ml (either RF-Ml3 or BlueScript). Samples were mixed on ice and used within 1 h of preparation. Specimens were warmed to 37 “C for approximately 3 to 5 min, ATP (or ATP@) was added to a concentration of 2 m&I, or as otherwise noted, and incubated for an addit’ional minute prior to application to electron microscope support grids. In most cases, 3 to 4 grids were prepared from each sample, over the course of approximately 1 min. Preparation of the final specimen was complete within 2 min after the addition of ATP. Samples (approx. 3 to 4 ~1) of the protein-DNA mixtures were applied t’o 300.mesh electron microscopy grids covered with carbon-support films, specially perforat,ed with numerous small (- 1 pm) holes. Samples were Slotted with filter paper and immediately plunged into a slurry of ethane cooled with liquid nitrogen, as previously described (Gogol et al.; 1989a,b), and transferred to liquid nitrogen for storage until examined in the electron microscope. Preparation of the frozen grids was carried out in a 4°C cold room to minimize evaporat.ion from the thin aqueous films and, in the latter stages of this work, within a partially enclosed hydrated chamber located within the cold room.

(f) Electron

microscopy

and image analysis

Specimens were examined in a Philips CM-12 electron microscope equipped with a Gatan cryo-holder and a locally designed and constructed double-blade anticontaminator. Images were recorded at a magnification of 57.000~ with minimal-dose methods on Kodak SO-163 film, developed in full-strength Kodak D19 developer for 12 min. The electron dose is estimated to be -20 e-/A”, Images were recorded with a defocus of approximately 0.9 pm. Areas of the micrographs chosen for image averaging were digitized with an Optronics PS-1000 rotating drum scanner? operated with a spot and raster size of 25 pm, translating to a specimen sampling pixel of 4.4 A (1 &!. = @l nm). Scans were converted to SPIDER (Frank et al., 1981a) format and displayed on a DEC microvax Y-GPX workstation as gray-level images, using locally written graphics programs. Individual particles were selected interactively, on the basis of clarity and nonoverlap with neighboring images, and windowed into indi-

vidual files composed of 32 by 32 pixeis. The files were scaled to give the same average (1.0) and standard deviation among pixels (@3). manReT, Image alignment was done in an iterative initially using a single image as reference. and subsequently averages of aligned images. The images were first Fourier transformed, filtered to exclude all information at resolutions iiner than 25 8; and back-transformed to produce low-pass-filtered images. These low-pass-filtered images were used to find optimal alignment parameters. both rotational and translational, by correlation t,o a single, clear, low-pass-filter image. These parameters were then applied to the initial (unfiltereclj images, and the of each image was judged both success of the a!ignment by visual inspection and by correlation to the reference image. 4 subset of well-aligned images was averaged a,nd used as a reference for further rounds of alignment. Yinally, all well-aligned images were averaged. including 7i% of the initial images. The resolution of t,he average was estimated by dividing the aligned images into 2 equal them separately. and subsets of images, averaging comparing the Fourier transforms of the 2 averages in radial increments. The resolut,ion was defined as the point, at which the incremental phase agreement between the two transforms equaled 45” (Frank et al.; 1981b). The aligned images were subjected to correspondence analysis to sort them objectively into categories that reflect the visible differences among them. The images Were each masked by a rectangular aperture to eliminate all features outside a box of 15 by 5 pixels (66 bv 22 A). centered on each image. This mask eliminated no&e peripheral to the hash-mark structures, as well as variability in the definition of the edges of the particles. The 120 images used were reduced to a set of Z-dimensional eigenfactors (which describe the major sources of variability

among the images) by the correspondence analysis option of the SPIDER programs. The meaning of each of the major factors was examined. and the relevant factors were identified that describe the variability in density at, the center of the images.

3. Results

(g44/62p and g45pj are added to RF-Ml3 DSA in the presence of g32p and ATP, distinctive structures associated witch the DNA become visible in cryoelectron migrographs (Fig. I). The DNA itself. in either the presence or absence of proteins, is readily visible in these images, at least in areas of thin unsupported films of vitreous ice (Fig. I (a)). The addit,ion of the four-pro&in mixture, under appropriate conditions, causes the appearance of short, (- 100 -4) bars (which we term “hash-marks” for their appearancej superimposed on some of the L>NA strands (Fig. l(b) and (c)J. These distinctive structures almost’ invariably cross the DKA strands in a direction perpendicular to the DSA axis, even on tightly curved DNA Most frequent’ly. the hashmarks are centered on the DNA, with very little asymmetry visible, above tha.t expected from the noise level. Visualization of these structures absolutely requires the presence of all three accessory proteins; the number of these structures is markedly increased (several-fold) by the addition of

Figure 1. Cryoelectron micrographs of unstained RF-Ml3 DNA embedded in amorphous ice, in (a) the absence and presence ((b) and (c)) of the 4-protein mixture, and of ATP. A few of the relatively isolated (non-clustered) hash-marks are indicated by black arrowheads in (b). (c) Examples of supercoiled DNA (white arrows), which are invariably found to lack any associated hash-mark structures. Inset: higher magnification of some clustered hash-marks on curved strands of DNA.

E. P. Qogol et a!

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g32p. Addition of g43p under conditions of DNA synthesis activity does not eliminate the hashmarks, although they are more dificult to see clearly because the DNA becomes quite aggregated and entangled (not shown). An absolute requirement for the formation of the hash-marks on DNA is the presence of ATP under conditions that permit the ATPase activity of the T4 DNA polymerase accessory proteins to function. No hash-marks are visible when ATP hydrolysis is prevented by chelation of magnesium ions by EDTA. Substitution of the ATP by the non-hydrolyzable analogue ATPyS also eliminates these structures. The hash-mark structures apparently have a limited lifetime, since depletion of ATP in the solution, as a consequence of the DNA-stimulated ATPase activity of the g44/62p complex, results in loss of these structure. (We estimate that the 2 mM-ATP in the mixture is depleted in -5 to 10 min at the concentrations of proteins and RF-Ml3 DNA used, on the basis of ATPase activity measurements conducted in our laboratory.) Interruption of ATPase activity by the addition of EDTA or dilution of the ATP with ATPyS also eliminates the hash-marks within one minute or less after addition of either of these reagents. The minimum time required for specimen preparation, from mixing to freezing, makes more precise measurement of the rate of hash-mark disappearance difficult. The hash-marks are occasionally visualized as isolated particles associated with the DNA strands (e.g. as indicated by some of the arrowheads in Fig. l(b) and (c)), but are more frequently seen in clusters along the DXA. The clusters are often up to -30 hash-marks (2000 to 3000 A) in length. Both clustered and relatively isolated hash-marks are often visible on the same strands of DNA, along with extensive lengths of DNA not, associated with any visible protein. The spacing of the hash-marks within a cluster can be as close as -70 8; but appears to be quite variable. The hash-mark structures were observed on many, but not all, of the observed strands of DNA. The specimen of RF-Ml3 DNA used in this study is primarily a mixture of native supercoiled DNA and DNA that has been relaxed by unknown mechanisms that may involve single or multiple nicks or gaps in the otherwise double-stranded DNA. Some of the DNA in the images has the intertwined appearance of supercoiled DNA (white arrows, Fig. 1(c)); these apparently supercoiled strands were never found to be decorated with hash-marks. This observation suggested that the physical state of the DNA platform (supercoiled or relaxed by nicks or single-stranded gaps) is important for the formation of the hash-mark structures. (b) Xtructures formed with plasmid

DNA

We next proceeded to examine the requirements for formation of the observed protein-DNA complexes using better-defined DNA cofactors. The

common cloning vector BlueScript was used for this purpose. This 2958 base-pair plasmid was prepared and isolated as an almost entirely supercoiled population of molecules (estimated as - 99 y0 supereoiled from ethidium bromide staining of agarose gels). The molecules differed in their state of concatamerization, ranging from monomers through tetramers. Occasionally trace amounts of even higher concatamers were observed. This plasmid was chosen because it contains the DNA sequence of the origin of replication of bacteriophage fl, a sequence that can be nicked at a single site with high efficiency by the fl gene II product (Meyer & Geider, 1979). Hence nearly uniform populations of supercoiled and nicked DNA molecules ea,n be produced and examined for their effectiveness as cofactors for the formation of the hash-mark structures. Nicks were introduced into supercoiled BlueScript preparations by incuba’cion with g2p, which converted from - 85 to - 99 y0 of the DNA, varying from preparation to preparation, to a rela.xed form. Agarose gels run in the presence of ethidium bromide indicated that (under the low Mg2+ conditions used) essentially all of the relaxed form remained nicked (RFIT) rather than reclosing to form un-nicked relaxed circles (RFIV), an activity demonstrated by the g2p under other salt eonditions (Meyer & Geider, 1979). The DNA molecules, regardless of their state of concatamerization: can be nicked at, only one site per molecule: as the g2p activity requires a supercoiled DNA substrate (Meyer & Geider, 1979). The uniqueness of the nick site was confirmed by restriction endonuclease digestion of the nicked DNA and electrophoresis under denaturing conditions, which yielded discret’e bands consistent with nicking at position 163 (data not shown). Single-stranded gaps were introduced int’o t,he relaxed closed circular plasmid DNA by 3’ to 5’ digestion of one strand from the unique nick site with exonuclease III. Specimens were incubated with exonuclease III as described in Materials and Methods. The non-processive nature of exonuelease XII digestion (Thomas & Olivera, 1978) and the high ratio of exonuclease III to DNA molecules should result in relatively synchronous digestion of 3’ ends (Wu et al., 1976; Guo & Wu, 1983). Thus the degree of digestion could be estimated by measuring the time required to reach the RsaI site at position 2526, 594 bases away in the 3’ to 5’ direction from the nick at position 163. At times shorter than that required to reach this site, all of the DNA was digested by Rsal to fragments of 1008 and 1870 base-pairs; at longer times, only the site at position 655 was cut by RsaI, and full-length fragments were therefore generated. These digestion times were measured and used to calculate the times necessary to produce approximately one-sixth or one-half this amount of single-stranded region (i.e. 5 or 15 min of incubation with g2p, to produce gaps of -100 or - 300 bases). When the supercoiled plasmid Dr\rA was substituted for the RF-Ml3 DNA used in the previous

T4 Accessory Protein-DNA

Complexes

401

Figure 2. Cryoelectron micrographs of plasmid DNA in the presence of the T4 accessory proteins (g44jSZp and g455 )I> relaxed by nicking by g2p; and (c) BlueScript with a 12~ and Mg-ATP: (a) supercoiled BlueScript; (b) BlueScript ogle-stranded gap of -300 bases. Arrowheads indicate examples of unclustered hash-marks present in (b) and (e).

402

8. P. Gogol

electron microscopy preparations, no hash-marks were found (Fig. 2(a)). The Dn’A moleclules displayed a highly supercoiled appearance in the micrographs, which was not visibly affected by the presence of the proteins in the mixture. Various lengths of molecules were seen, consistent with the range of concatamers in the preparations, though individual molecules were difficult to separate and visually trace in more crowded fields of view. The nicked DNA had lost the supercoiled appearance of the unmodified plasmid, and appeared as relaxed loops (Fig. 2(b)). The size and uneven distribution of the DNA loops over the holes in the carbon film made tracing their contours difficult, except for occasional molecules that were very fortuitously displayed. As noted with the supercoiled plasmid, defining the separate paths of nearby or overlapped molecules usually proved difficult. We attempted to locate the nick in the molecules by binding T7 RNA polymerase to a nearby T7 promoter. We failed to visualize the T7 polymerase as an identifiable structure bound to DNA, perhaps because conditions used were not ideal for the formation and stability of open promoter complexes. Addition of the accessory proteins plus g32p and ATP resulted in the appearance of hash-marks on occasional DNA strands, both in clusters and individually or in small groups (Fig. 2(b)). In contrast to the RF-Ml3 DNA, however, relatively few hash-marks were observed in most fields of view. Though nearly all of the DNA strands were nicked, most of them were not adorned with hash-marks. In the absence of g32p, even fewer of these structures were seen on the nicked DNA. The gapped DNA proved to be a much better substrate for the formation of hash-marks. Fields of view were often crowded with clusters of hashmarks (Fig. 2(c)), which occasionally covered a much greater length of DNA than the estimated 100 or 300-nncleotide gap. Direct verification of the gap size was difficult, since no distinctive features were present to identify the location of the gaps in any of the micrographs. The conditions for formation of the hash-marks were identical to those determined for RF-Ml3 DNA: the presence of the accessory proteins and ATP hydrolysis were required to obtain the structures. Addition of g32p noticeably increased the number of hash-marks visible in fields of view. When ATP hydrolysis was prevented (by of ATPyS), or lack of ATP or Mg2 ‘, or substitution either the g44/62p or g45p complex was omitted, the hash-marks failed to appear. (c) Linear

DNA

cofactors

Linear DNA fragments were made from intact supercoiled, nicked and gapped plasmid DNA by digestion with RsaI, yielding two blunt-end fragments, of lengths 1008 and 1870 base-pairs. The shorter fragment contains the origin site (nick or 3 end of the gap if prepared from modified plasmids) approximately at its center. Since a large fraction of the plasmid was concatamerized, and only one site

et al.

was nicked per plasmid, the 1008 base-pair fragment is a mixture of modified (nicked or gapped) and intact linear DNA. Agarose gels revealed that all the DNA visible by ethidium bromide staining was in the form of these two linear fragments. The two fragments were not, separated, but were used as a mixture for electron microscopic experiments. When intact linear fragments were mixed with the proteins and ATP, hash-marks were not visualized in associat,ion with the DNA (Fig. 3(a)). Preparations containing the nicked fragments only rarely displayed hash-marks, and those few hashmarks that were seen were usually isolated single structures (Fig. 3(b)), though small clusters were even more rarely noted (Fig. 3(c)). Mixt,ures containing fragments of the gapped plasmid more frequent,ly resulted in fields of view containing hashmarks, often in small clusters (Fig. 3(d) and (e)). With both nicked and gapped linear fragment preparations, qualitatively fewer structures were observed in associat,ion with the DNA than were seen with the circular plasmids. Since the DNA fragments in these preparations were not separated, the 1870 base-pair fragment, not containing the nick site comprises half (by number) of the strands visualized; by length, this fragment con’tains almost two-thirds of the DNA in the preparations. Furthermore, since the plasmid DKA is a mixture of monomers and concatamers, and only one nick is made on any molecule by the g2p (supercoiled DNA is required for nicking), many of the 1008 base-pair fragments do not contain a nick or gap. Even in the best preparations, the number of intact DNA fragments far exceeds that of the nicked or gapped DNA fragments. This is in contrast to the situation in the circular plasmid preparations, in which almost all molecules are modified. (d) Stimulation

of the g44j62p

ATPase

activity

The various forms of plasmid DNA4 were examined for their ability to act as cofactors for the DNA-stimulated ATPase activity of the g44/62p complex. While the supercoiled BlueScriptj showed virtually no stimulation of the ATPase activity over that inherent in the prot’ein preparations alone (Fig. 4), the nicked and gapped molecules (from the same preparation of plasmid) stimulated the ATPase in a concentration-dependent way. Gapped DNA with t,wo different-sized single-stranded regions (N 100 and -300 bases) was used, and the extent of ATPase stimulation was comparable in both, at a level approximately twice that observed with a singly nicked DNA. The data in Figure 4 are plotted as a function of the weight concentrat’ion of DNA; since the DNA is a mixture of concatamers, it is difficult to estimate the molar coneent,ration accurately. However, since all the data shown were collected using a single preparation of plasmids, the same distribution of concatamers is present in all forms. Although hash-marks are highly clustered in most eases, the ATPase activity shows a strong DNA concentration dependence. This is due to the

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Fi gure 3. Cryoelectron micrographs of linear fragments of BlueScript plus the protein-ATP mixture. Fragme nts of: (a) intact (supercoiled) plasmid; (b) and (c) singly nicked plasmid; (d) and (e) prep ared by limited RsaI digestion bases) plasmid. Individual hash-marks in (b) and (e) are indicated by arrowheads, as is the small cluf ker izap5 )ed (-300 in (c ).

E. P. Gogoi et ai.

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L .P E 3 -E P

Gapped t-300 bases) Gapped(-100 bases)

4

I 0 t

2

?

Suoercoiled DNA

[DNA]

I

(W/ml)

Figure 4. Stimulation of the ATPase activity of the T4 accessory proteins complex by various forms of plasmid DNA.

fact that the DNA concentration range used in the kinetics experiments is in the range of the K,,, for DNA of the accessory proteins (lo-’ M), and therefore the dependence on DNA concentration reflects the association of the first accessory complex. The ATPase stimulation experiments were reproduced with severa, different DNA preparations with comparable results, differing only in the quantitative degree of stimulation among preparations with different concatamer distributions, or different efhciencies of nicking. ATPase stimulation was also measured in the absence of g32p. The results are similar to those displayed in Figure 4, but the activity levels were only half those measured in the presence of 1 PM-g32p.

(e) Image averaging Most of the hash-marks visible on the DNA strands were of approximately the same length, and usually oriented with their long dimensions perpendicular to the DNA. Only very occasionally were particles observed that were oriented at an angle of -45” with respect to the DNA (not shown). These rare particles were excluded for the purposes of image analysis and averaging, and only the majority images, of approximately equal dimensions, with perpendicular orientation to the DNA and reasonably good definition (without overlap with neighboring or underlying structures), were selected. Of the 156 images initially selected, the alignment procedure described in Materials and Methods resulted in an apparently correct alignment of 120 images. The initial reference image chosen for this alignment was rotated to place the DNA strand vertically, yielding the averaged image shown in Figure 5. The resolution of this image, as defined in Materials and Methods, is approximately

30 A. The two-dimensional size of the hash-mark structures can be easily estimated from the averaged length and -30 A in image, as -90 A maximum width. The recorded images are projections through

I

- 50,000daltons

- 150,000daltons

Figure 5. (a) An average of 120 aligned images of hashmark structures. The images have been aligned to place the DNA vertically in this Figure. (b) Cylindrical models to represent the 3-dimensional size of the st,ructures. along with their corresponding equivalent masses (as protein).

the structure and do not contain information about the dimension in the direction normal to t’he plane of the image. However, we can make reasonable models of the structure in three dimensions. Figure 5 shows two simple cylindrical models of the structures, one t,hin and narrow and the other wide and flat. For the narrow cylinder, the uniform size of the structures seen in the electron microgr,aphs requires a specific interaction that would eonsistently orient these structures in the plane of the ice film. In contrast, the flat cylinder has identical dimensions from any viewing direction perpendicular to the axis of the DKA molecule, which runs through its center. The equivalent protein molecular weights for these simple models, derived from their volumes, indicate that the narrow cylinder could accommodate only -50 kDa of protein mass, while the flat model could accommodate - 150 kDa, equivalent to approximately one g44/62p (4 : 1) complex (Jarvis et aZ., 1989a). Similar size estimates are obtained by modeling the structures as prolate and oblate ellipsoids.

(f) Substructure and image classification Inspection of the aligned images, particularly in low-pass-filtered form, revealed a readily visibie source of structural variability among the images. This difference is the density at the center of the particle, i.e. the intersection of the DNA with the

T4 Accessory Protein-DNA

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Figure 6. Low-pass-filtered images of examples of hash-marks with low-density centres (top row) and those with highdensity centers (bottom row). Images were Fourier-filtered at a resolution of 25 a to reduce the high-resolution noise in the images, and were selected on the basis of the clarity of their substructures.

hash-mark structure. In many of the images the center represents a density minimum within the particle, while in others it is a maximum, often even representing the highest density in the particle. Examples of individual low-pass-filtered images of each category are shown in Figure 6. Images of both types are found associated with the same DNA molecules, often next to one another, and display no visible differences in size. Many of the images are also not readily classified into these two categories, lacking visible substructure along their lengths. The variability in the images was examined by correspondence analysis, a method of reducing the major sources of variability in a population of images into a basic set of objectively defined eigenfactors (van Heel & Frank, 1981; Bretaudiere & Frank, 1986). Each of these eigenfactors is a twodimensional eigenfunction that describes a major variation among the images in the set. Thus each image can be reconstituted as a linear combination of the average image and the eigenfactors, each weighted by the eigenvalue appropriate for that image. For this analysis, each of the 120 aligned images was masked to eliminate the peripheries and surrounding backgrounds of each image and the correspondence analysis procedure, as incorporated in SPIDER, was applied. This procedure yielded a set of two-dimensional eigenfactors, whose meanings were determined by calculating their individual influences on the averaged image. Examples of these reconstituted one-factor images are shown as insets at the edges of Figure 7. Some of the largest eigenfactors corresponded to slight tilts of the images, or to left-right asymmetries (not shown). These are most likely due to noise in the images, or to imperfect alignment of some images. Among the largest eigenfactors (those describing major sources of variation among many of the images), two were consistently found that define an internal variation in the particle density. This variation is similar to that noted visually; that is, a difference in density at the center of the particle. Images reconstituted from the extremes of both eigenfactors are shown as insets in the upper right and lower left corners of Figure 7. With the relatively small mask dimensions used in the analysis shown in Figure 7, the relevant

eigenfactors were the second and fourth in size. When the analysis was repeated using less-restrictive masks, these same eigenfactors consistently appeared among the four largest, demonstrating the importance of this structural variation among these images. The distribution of the images according to the two factors that describe this internal variation among the hash-marks is shown in Figure 7, in which the position of each image (shown as an asterisk) is determined by the corresponding value of the two relevant factors. No clustering of images into easily defined sets is readily visible. The distri-

Figure 7. Plot of eigenfactors obtained by correspondence analysis of the 120 aligned images used in Fig. 5. Images at the centers of the edges of the plot are reconstituted from the extreme values of factors 2 and 4, and demonstrate the variability represented by these factors; those at the upper right and lower left corner are reconstituted from the combination of the 2 factors. The diagonal lines drawn on the Figure are those used to divide the apparently continuously distributed population of images into 3 equal-sized categories.

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Figure 8. Averages of 40 images each selected from the 120 aligned images of the hash-mark structures by the correspondence analysis eigenfactor map shown in Fig. 6.

bution is in fact continuous, suggesting that the observed variability in the feature described by these factors (i.e. the density at the center of the hash-marks) is a continuum among the images. Such a distribution may be the result of a continuous variation in viewing angles around a structure. Since no clear way of separating the images into classes was obvious, they were divided into three equal-sized categories by straight diagonal lines drawn across the map of the two factors (Fig. 7). Three categories, representing high and low-density centers, were also suggested by visual inspection of the images. The three classes, each containing 40 images, were individually averaged, resulting in the images shown in Figure 8. Resolution estimates calculated for each of the three averages are in the range 30 to 35 A.

4. Discussion microscopy for the study of protein-DNA complexes

(a) Cryoetectron

The distinctive protein-DNA structures described in this study have been visualized under conditions similar to those required for the activation of the T4 DNA-dependent DN-4 polymerase by the T4 accessory proteins. These include the presence of the three accessory proteins (g44/62p complex and g45p) and ATP hydrolysis induced by their DNA-stimulated ATPase activity. The frequency of the appearance of these structures in association with nicked and gapped cofactors is

increased by the addition of g32p, which causes a similar stimulation of DNA synthesis by the T4 polymerase and accessory proteins (Huberman et al., 1971; Huang et al., 1981). These conditions have been preserved, with no addition of chemical agents, or dilution of buffers or macromolecular components, by examination of amorphously frozen specimens. Since the structures are labile to chemical disruption (e.g. by interrupting ATP hydrolysis with EDTA) or ATP depletion as a consequence of t,he ATPase act,ivit,y of the g44/62p, it is vital to isolate and trap these assembhes without disruption. The very rapid freezing process employed accomplishes t,his. By not adding any agents to increase the electron-scattering density of the specimen, the contrast in t’he images is limited to that corresponding t,o the relative scattering densities of protein, DNA and the aqueous buffer. Nevertheless, in areas of s&iciently thin ice; adequate contrast is achieved to visualize easily both naked DNA and protein complexes in association with the DNA. However, the contrast is considerably lower than that available with heavy-metal shadowing of DNA and, except in the most favorable examples, it is rather difficult to follow the path of individual DNA molecules for their entire lengths. Tracing the DNA molecules is further complicated by the threedimensional nature of the specimen: rather than being spread on a t’wo-dimensional surface, the molecules are free to distribute themselves in t,he thin t’hree-dimensional layer of solution prior to freezing. The large dept,h of field in the electron microscope (-1oooA under the conditions employed) ensures that the DNA, and the associated protein st,ructures, are recorded in full projection rather t,han being optically sectioned. By eliminating the use of metal casts, the outlines of the complexes more accurately reflect the true projections of the structures, rather than surface topology as defined by the deposition of metal grains. Thus the possibility exists of gaining information about the internal structure of the DNA-protein complex. Furthermore, the possibility of struct’ural distortions resulting from adsorption onto solid surfaces is greatly reduced, as only those molecules not in contact with any support are examined. (b) Sfructure

oj’ the accessory prote*in-Dil’A

compir~en

The distinctive hash-marks formed by the T4 rephcation accessory proteins in association with DNA makes their identification very easy even in the relatively low-contrast unstained images presented in this paper. Most of the structures identified display a striking similarity in size and shape, i.e. they appear as elongated bars approximately 90 L% long (across the DNA) and 30 A wide. Very simply modeled in three dimensions (as in Fig. 5), these complexes are consistent with molecular volumes of 65,000 to 190,000 A3, or protein mokcular masses of -50 to 150 kDa. Distinguishing

T4 Accessory

Protein-DNA

between these two extremes of reasonable size estimates requires information on the third dimension of these projection images. The uniform appearance of the protein-DNA assemblies suggests that some degree of orientation is imposed on the complexes. Owing to their size and fibrous nature, the long strands of DNA with may be are associated which the proteins constrained to lie primarily in the plane of the ice film. This restriction is expected to limit the variability of views of the complexes mainly to rotations around the DNA axis. In the absence of specific orientation effects at the water surface, and since no solid substratum is present (over the holes) to which the samples can adsorb, a continuous angular range of projections of the protein complexes about the DNA axis is expected. The consistency seen in the dimensions of the particles supports the argument for a three-dimensional structure that is relatively invariant around the DNA axis, such as an oblate ellipsoid with the DNA passing through its center. However, preferred orientations of macromolecular complexes can be introduced, presumably by fortuitous interactions of proteins with the air-water interfaces of the thin aqueous films, as has been noted with several specimens examined by cryoelectron microscopy (Dubochet et al., 1988; Gogol et al., 1989b, 1991). If the accessory protein complexes exhibit a preferred interaction with the air-water interface immediately before the samples are frozen, the resulting range of views might be limited, and even a very asymmetric structure (like a prolate ellipsoid) may be seen in only an extended projection. The two simple structural models shown in Figure 5 give very different estimates of the size of the complex, which set different limits on the compositions of the hash-mark structures. The hashmarks are visualized only in the presence of the g44/ 62p and g45p complexes, which have molecular masses of - 160 kDa and -74 kDa, respectively, as oligomers in solution (4 : 1 complex of g44/62p, and trimers of g45p; Jarvis et al., 1989a). The smaller, prolate model of the hash-marks is too small to accommodate either of these protein complexes. The larger oblate model, on the other hand, is approximately the size of the g44/62p complex, and twice the size of the g45p trimer. Since the g44/62p and g45p subassemblies each form separate, very tight complexes in solution (Jarvis et al., 1990, 1991), it is likely that their stoichiometries are preserved when bound to DNA. Consideration of the size of the subassemblies suggests that the hash-mark structures are made up of one g44/62p complex, or one or two g45p trimers, arranged as a flat structure pierced by the DNA axis. The observation by Jarvis et al. (1990) of an optimal stoichiometry of one 4 : 1 complex of g44/62p to one g45p trimer in ATPase assays argues against the formation of dimers of the g45p trimers. Examination of g44/62p and g45p subassemblies in the absence of DNA reveals a variety of projection structures (not shown), indicating that no

Complexes

407

single orientation of the proteins in the aqueous film is preferred when they are not associated with DNA. Among the various projections that each of these two protein complexes display are a small number of narrow linear views, similar in size to the hashmarks visualized with DNA. Hence it is not possible to identify either one of these isolated protein complexes uniquely with the hash-marks.

(c) Substructure

of the assemblies

The substructure visible in the images of the hash-marks provides further information on the arrangement of subunits within the structures. Visual examination of individual images readily identifies differences in the modulation of density along the lengths of the hash-marks. Correspondence analysis independently and more objectively reveals these differences to be major sources of the variation among the images examined. (Other major differences appear to be due to imperfections in alignment of all the images, and to noise in the micrographs.) Two distinct classes of images have been identified, those comprising two high-density regions separated by a low-density region at the position of the DNA axis, and those containing three high-density regions, including one superimposed on the DNA axis. The separation of the images by correspondence analysis appears to be a continuum with respect to the variation of this feature, with images between the two extremes displaying little or no lengthwise modulation of density. A simple interpretation of this variation among the images is that they are different projections of a single structure, rotated about the DNA axis passing through its center. An alternative explanation would be that there are two or more classes of hash-mark complexes, with different compositions or structural arrangements. The highdensity regions in the images are the result of superposition of protein subunits that make up the structure, and two simple arrangements that explain the projections obtained are shown in Figure 9. In one case (top of Fig. 9), a planar tetragon of subunits, viewed edge-on, results in the three general classes of views (center row). A triangular arrangement of subunits (bottom of Fig. 9) can also lead to a similar set of projections. The stoichiometry of the subunits in the tetragonal model suggests that they may be monomers of g44p; the trigonal model likewise suggests the composition to be g45p monomers. In each model, the ellipsoids predict monomer volumes of the molecular masses that are close to those of the proteins: 34 kDa for the tetramer (cf. 25 kDa for g44p) and 24 kDa for the trimer (cf. 31 kDa for g45p). On the basis of the projection views alone, it is not possible to determine the composition of the hash-mark structures. It is even possible that the hash-marks may be composed of all three proteins in a stoichiometry different from that of the two subcomplexes in solution. This last possibility seems

408

E. P. QogoE et al.

Figure 9. Two simple models of the hash-mark structure as a planar tetramer of subunits (top left); or as a planar trimer (bottom left); which can explain the different projections shown in Fig. 8. Top views of the models (top right and bottom right) indicate various viewing directions that would result in projections A, B and C, drawn schematically in the middle of the Figure.

unlikely in view of the great stability of both the g44/62p and g45p complexes (Jarvis, 1989a). A number of other observations and considerations are relevant to the question of the composition of the hash-mark structures. Photochemical crosslinking of the accessory protein complex to DNA suggests that, in the assembled complex, the g62p is the subunit most intimately associated with et al., 1987; Kubasek et aZ., DNA (Hockensmith 1989; Capson et al.; 1991), and hence may be at the center of the structure. If the hash-marks are g44/ 62p complexes, as specified by the tetragonal model, the g62p is not evident. Location of the g62p at the centre of a tetragonal g44p complex might explain its invisibility in projection, since the g44p subunits and the DNA itself would tend to obscure the smaller g62p. All three proteins are required to obtain the hash-marks, so at some point both the g44/62p and g45p complexes most probably associate with the DNA; the hash-marks may simply be the majority population, or may be a mixture of two compositionally different, though structurally similar, complexes. Questions are also raised by the observed shape of the hash-mark structures. Previous characterization of the isolated g44/62p and g45p complexes by hydrodynamic methods has suggested that they both have highly asymmetric shapes, such as prolate ellipsoids with axial ratios of N 5 : 1 (Jarvis et al., 1989a). Modeled as oblate ellipsoids, the axial

ratios would be even larger (-5.5). These estimates assume a degree of hydration in the “average” range of 93 to 0.4 g of water per gram of prot,ein, and may be reduced if the protein complexes are more heavily hydrated. The DNA-associated strucLures t,hat we have visualized do not exhibit such a high asymmetry, either modeled as a cylinder of an ellipsoid ( -3 : 1 axial ratio), or as a square planar or trigonal arrangement of nearly spherical subunits (Pig. 9). This discrepancy could be due to: (I) a bigher than expected degree of hydration of the complexes; (2) additional parts of the hash-mark structures not visualized in the averaged images, perhaps due to conformational variability; or (3) macromolecular reorganization of the complex on binding to DNA. While it is not, possible to discount the first, two possibilities, at least a transient rearrangement of subunits of the free complex seems necessary to accommodate the association of a continuous DNA strand through the center of the protein complex. This redistribution may indeed be as extreme as a change from an open, even linear: arrangement of subunits (e.g. g44p or g45p monomers) into a compact, planar tetramer or trimer. Owing to the unstable nature of the proteinDNA complex, it is not possible to make a direct comparison of the hydrodynamic properties of the free and DNA-bound complexes. It has been di%cult to interpret electron micrographs of either the g44/62p or g45p complexes, owing to the multiplicity of projections visualized, but approaches to examining these in more detail are currently being developed. (d) DLNA cofactor

requirements

The hash-mark complexes have been visualized in association with only a subset of DNA cofactors. This selectivity was first noted in the mixed population of RF-Ml3 DNA molecules, where hash-marks are never seen on DNA that appears supercoiled, while apparently relaxed DPLA in the same field of view is often heavily coated with the structures. The cofactor requirements were examined by using plasmid DNA, which was prepared in three different physical states: (1) supercoiled; (2) nicked at a single site; and (3) gapped with a short single-stranded region. The requirement for nicks (or gaps) in the DNA to form the structures was supported by these experiments, in which hash-marks were never seen with supercoiled plasmid DNA, but were present when the only modification to the DEA was the addition of a single nick. The gapped plasmid DNA proved to be an even better cofactor, as the frequency of hash-mark appearance was significantly increased over that seen with nicked DNA. Qwing to the large variability in the number and distribution of these st’ructures (as well as DNA molecules), even among fields of a single specimen, it is difficult to make a quantitative estimate of the difference between nicked and gapped DNA as cofactors. However, the frequency of encountering bash-marks appears markedly higher, perhaps five-

T4 Accessory Protein-DNA to tenfold, among several different preparations examined. The experiments done with linear DNA fragments of intact, nicked and gapped plasmids demonstrate that relief from the physical constraints of supercoiling is not suliiicient for formation of hash-marks on otherwise intact double-stranded DNA. Likewise, the blunt DNA ends of the fragments are not adequate cofactors for assembly of the proteinDNA complexes. Nicks or gaps in the DNA are necessary, with gaps once again the better cofactor, judged by the much greater number of hash-marks seen. Single-stranded gaps in double-stranded DNA are analogous to the primer-template junction of replicating DNA, differing mainly in the lack of an open fork in the DNA. It is known that the accessory proteins preferentially associate with primertemplate junctions (Munn, 1986; Jarvis et aE., 19893; Kubasek et al., 1989), but the single-stranded DNA requirements of the complex on the primertemplate are not precisely known. If hash-marks require a single-stranded region to associate with DNA, they would be expected to form more frequently with gapped than with nicked cofactors; this preference is indeed observed with the modified plasmid cofactors. The finding that hash-marks are able to form on nicked cofactors suggests either that the accessory proteins can assemble at nicked structures, perhaps with lower efficiency, or that small single-stranded structures are formed at the nicks by separation of a short segment of DNA (5’ to the nick) from its complementary strand. Fraying of the ends of the DNA is a transient and rare event, rather than a“‘permanent” structure like the singlestranded regions in a gapped DNA molecule. By binding the transiently single-stranded regions at the frayed ends of nicks, g32p can increase the lifetime of these opened structures and thus stimulate the formation of the hash-mark complexes. This protein can also play a similar role in inhibiting the formation of secondary structure in single-stranded gaps in DNA. The DNA-stimulated ATPase activity of the accessory proteins shows a preference for DNA cofactors that parallels that for the formation of the hash-marks. Supercoiled plasmid DNA effects no stimulation of the ATPase, but nicked and gapped DNA were activating cofactors, differing by a factor of approximately 2 in their effectiveness. As the ATPase activity is necessary for the enhancement of DNA polymerase (g43p) activity by the proteins, this enzymatic difference among DNA cofactors is a link between the function of the accessory proteins and the appearance of the structures visualized in the electron micrographs. (e) Clustering

of the

hash-marks

The distribution of hash-marks on DNA cofactors in most fields of view includes clusters of the structures, containing from two to more than 25 of the complexes. Though single isolated hash-marks are

Complexes

469

Figure 10. A model for the assembly of hash-mark structures at nicks or gaps in double-stranded DKA is illustrated schematically. Migration of the assemblies away from their points of initial association with the DNA leads to clusters on one or perhaps both sides of their assembly points.

also invariably present, the clustered ones usually account for the majority of those seen. Clusters of hash-marks are present even with the singly nicked plasmid DNA cofactors, though both single hashmarks and clusters are more frequently encountered on DNA with single-stranded gaps. This uneven distribution of the structures suggests some sort of co-operativity in their formation, or self-association following assembly on DNA. The spacing of hashmarks within the clusters is rather variable, with the closest being -70 A in center-to-center distance. The uneven spacing argues against direct protein-protein interactions between neighboring hash-marks as the driving force behind the clustering, since similar contacts could not be maintained over the range of distances seen. As the presence of nicks or gaps in the DNA appears to be critical for the formation of hashmarks, it is reasonable to suggest that the assemblies may form at these interruptions in the doublestranded DNA. To function as an assembly site, the DNA may need to undergo some transient rearrangement, such as fraying of the doublestranded end. As schematically suggested in Figure 10, these assembly sites may be the point of injection of the structures into the double-stranded DNA, and thus form the basis of the clustering of hash-marks. The presence of g32p, which promotes

E. P. Qogol et al.

410

but is not required for formation of bash-marks, would stabilize a partially open DNA structure like the postulated one. If the DNA-associated complexes are free to diffuse along the DNA strands following their assembly, they will migrate (in one or both directions) away from their initial assembly points, freeing these sites for association with other protein complexes. The size of the observed clusters suggests that bash-mark formation is co-operative. These experiments were carried out at protein to DNA ratios of -5 g44/62p complexes, 50 g45p trimers and 120 g32p monomers per DNA molecule. If the hash-marks are indeed g44/62p complexes, then the average number per DNA molecule, either clustered or dispersed, should be five. Even if the hash-marks are composed of g45p, the number of g44/62p complexes might be expected to limit the size of the clusters. Since the clusters on average contain well over five hash-marks, the association of one hash-mark with a DNA molecule appears to facilitate the formation of subsequent hash-marks at that site. This might result from the persistence of some special DNA arrangement after the newly formed hash-mark migrates away, providing a nucleation site for formation of further complexes. In this case: the “used” site may be favored over other nicks or gaps, since it has a DNA configuration ready for assembly of another hash-mark. Depending on the relative rates of hash-mark formation, migration along the DXA and disassembly, the complexes may “pile up” at one or both sides of the assembly points. The average size of t,he resulting clusters would depend on the ratio of these rates, as well as the lifetime of the nick/gap as an active assembly point. Few clusters are noted on the linear DNA cofactors. This observation may be due to migration of the hash-marks off the ends of the fragments, as this distance is only -500 base-pairs from the nick site in either direction. In the case of gapped fragments, the 3’ ends of the gaps are even closer to the ends of the fragments. In contrast to the closed circular DNA cofactors, the lifetimes of the associated hashmarks may depend on the migration rates along the DNA, rather than the rate of disassembly of the complex. (f) Functional

signi$cance

The conditions necessary to form and visualize the hash-marks are similar to those required for the stimulation of the processivity and rate of the g43p DNA polymerase. It is likely that these structures comprise at least part of the complex that associates with g43p to form the active polymerase holoenzyme. The role of the accessory proteins in enhancing polymerase processivity has been conclamp” or “platform” that sidered as a “sliding stabilizes the holoenzyme at the primer-template junction within a replication fork (Newport et al., 1980; Huang et al., 1981; Munn, 1986; Jarvis et al., 1991). We have shown that, a primer-template junction analogue (a nick or gap in double-stranded

DKA) is required to form the hash-mark strractures, and have suggested that these assemblies may associate with the DNA at these points. These struct(ures have a finite lifetime; as demonstrated by their disappearance within minutes of stopping ATP hydrolysis. A recurrent’ rather than continuous ATP requirement has been shown for the stimulation of polymerase processivity (Jarvis et al., 1991), which would be consistent with an ATP requirement for the formation of hash-marks, with the resulting protein-DNA assemblies displaying a finite lifetime. The postulated migra,tion of the hash-marks along the DNA at first appears to be inconsistent wit*h the putative role of the accessory proteins in keeping the holoenzyme at the primer-template junction. However, the structures visualized do not, contain the full complement of proteins of the holoenzyme, particularly as the polymerase (g43p) is not present. Furthermore, the size of the structure suggests that both g44/62p and g45p cannot be accommodated, if their free solution stoichiometries are to be maintained. The complete holoenzyme complex may not exhibit, the ability to migrate along the DNA, which has been suggested for the bash-marks. However, we have shown that inclusion of g43p in the protein mixture (at least when added in substoichiometrie quantities) does not entirely prevent the visualization of hash-marks. The T4 accessory proteins have been shown to stimulate the transcription of late T4 genes by the T4-modified E. coli DKA-dependent RXA poly merase (Herendeen et al., 1989). In vitro, t’he stimulation of transcription requires nicked doublestranded DNA templates, and t’he affected promoters can be at least hundreds of base-pairs distant, in either direction, from the site of the nick. Neither relaxed, covalently closed circular DSA nor intact linearized fragments exhibit this t,ranseription stimulation. The observed stimulation, like the enhancement of DNA synthesis, is dependent on XTP hydrolysis, and is inhibited by ATPyS. The proposed role of the nick in these assays is in t’he binding of the g44/62p and g45p, followed by migration of the bound proteins bidirect,ionally along the DKA to act as stimulatory factors that are functionally specific for T4 late promoters. In a further analogy to the requirements for the formation of the hash-marks, the activation of the RNA polymerase does not require g32p, but is stimulated by its presence. The requirements for formation of hashmark assemblies and the properties inferred from their observed distribution appear to fit th.ose observed for the stimulation of transcription, and the structures identified in this study are likely to be identical with those that have been shown to stimulate transcription from T4 late promoters in the studies of Herendeen et aE. (1989: 1990). This research was supported, in part, by USPHS Research grants GM-15792 and GM-29158 (to P.H.von H.), by an institutional grant from the Lucille P. Markey Charitable Trust, by USPHS Individual Postdoctoral R’ational Research Service Sward 81-07568

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(to M.C.Y.) and by traineeships on USPHS Institutional Service Award GM-07759 (to W.L.K. and T.C.J.). P.H.von H. is an American Cancer Society Research Professor of Chemistry. We are very grateful to Dr Nick Davis for suggesting and advising on the use of g2p, to Dr Michael Reddy for his advice and comments throughout this work, and Dennis Spaan, Steven Weitzel and Jim Linn for their technical assistance. We would also like to thank Drs E. P. Geiduschek and Bruce Alberts for their helpful comments on the manuscript.

(1989a). Molecular architecture of Escherichia coli F, adenosinetriphosphatase. Biochemistry, 28, 47094716. Gogol, E. P., Aggeler, R., Sagermann, M. & Capaldi, R. A. (19896). Cryoelectron microscopy of Escherichia coli F, adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex. Biochemistry, 28, 4717-4724. Gogol, E. P., Johnson, E., Aggeler, R. & Capaldi, R. A. (1990). Ligand-dependent structural variations in Escherichia coZi Fr ATPase revealed by cryoelectron microscopy. Proc. Nat. Acud. Sci., U.S.A. 87, 9585-

References

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by R. Huber