©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Escherichia coli DNA Polymerase III Holoenzyme Subunits , , and Directly Contact the Primer-Template (*)

(Received for publication, November 2, 1994; and in revised form, December 16, 1994)

Jo Anna Reems (§) Steve Wood (¶) Charles S. McHenry (**)

From the Department of Biochemistry, Biophysics, and Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the alpha, beta, and subunits formed ATP-dependent contacts with the primer-template. The alpha polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The subunit contacted the primer at positions -13, -18, and -22, with the strongest -primer interactions occurring at position -18. The beta subunit predominated in cross-linking at position -22. Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3`-primer terminus followed by at -18 and beta at -22, and the subunit remains a part of the initiation complex, bridging the alpha and beta subunits.

Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (-complex (--`--) and beta subunit) revealed the subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of by alpha at the primer terminus. These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.


INTRODUCTION

The multisubunit enzyme DNA polymerase III holoenzyme (holoenzyme) (^1)is the major replicative enzyme responsible for the synthesis of the Escherichia coli chromosome. This enzyme is comprised of at least 10 different subunits (alpha, , beta, , , , `, , , and ) (McHenry and Kornberg, 1977; McHenry and Crow, 1979; McHenry, 1982, 1988; McHenry et al., 1986; Maki and Kornberg, 1988a; O'Donnell and Studwell, 1990). Due to the tendency of holoenzyme to dissociate during the purification process, three functionally distinct subassembly forms, Pol III, Pol III`, and core Pol III, have been isolated. The tightly associated alpha, , and subunits comprise the simplest holoenzyme form, core DNA Pol III. The alpha and subunits possess the catalytic sites for the polymerization and 3`5` exonuclease activities, respectively, while the function of the subunit remains unclear (Slater et al., 1994). The association of the auxiliary subunits with core Pol III defines the subassemblies Pol III` (core Pol III plus the subunit) and Pol III* (holoenzyme minus the beta subunit).

The effects of ATP, SSB, and salt upon DNA polymerase III are influenced by the presence or absence of the auxiliary subunits. A distinction between holoenzyme and its simpler subassembly forms is the ability of holoenzyme to form a stable initiation complex with a primed template at the expense of ATP hydrolysis. The auxiliary subunits act to lock the core polymerase onto a primer-template in an ATP (or dATP)-dependent reaction creating a complex capable of replicating a natural chromosome in a rapid and highly processive manner (Wickner and Kornberg, 1973; Wickner, 1976; Johanson and McHenry, 1982; Burgers and Kornberg, 1982). In the absence of the auxiliary subunits, ATP is ineffective in inducing the formation of stable complexes (Johanson and McHenry, 1982) dramatically decreasing processivity. Both the polymerase and 3`5` exonuclease activities are inhibited by high salt concentrations and SSB (Fay et al., 1981, 1982; Mok and Marians, 1987; Griep and McHenry, 1989; Reems et al., 1991) in the absence of auxiliary subunits. Thus, the auxiliary subunits appear to reverse these inhibitory effects in the simpler polymerase forms.

In vitro, holoenzyme can be loaded onto the primer-template in two distinct stages. In the first stage (preinitiation complex formation), the -complex (, , `, , and subunits) directs the beta subunit to the initiation site in an ATP-dependent reaction (Wickner, 1976; O'Donnell, 1987). In the second stage, an initiation complex is formed by the addition of core DNA polymerase III to the preinitiation complex creating a complex capable of rapid and highly processive DNA replication (O'Donnell, 1987).

The availability of special photo-activatible probes strategically positioned within a primer-template provides a means to determine protein-DNA contact points in a site-specific manner. One such system designed by Gibson and Benkovic(1987) has been used to identify the molecular interactions within the DNA polymerase I-primer-template complex and between T4 DNA polymerase holoenzyme and its primer-template (Catalano et al., 1990; Capson et al., 1991) The T4 replicative complex, also a multienzyme system, contains the polymerase gene 43 and its associated ancillary subunits, genes 44/62 and 45. Capson and colleagues(1991) determined that within the T4 initiation complex, gene 43 contacted position -4 upstream of the 3`-primer terminus followed by gene 62 at -9 and gene 45 at -14-20.

In this study we have used the same nitrophenyl azide probes developed by Benkovic to identify E. coli DNA Pol III holoenzyme contacts with primer-template. Using a series of seven photo-activatible primers annealed to a derivative of M13mp19, we found that within the initiation complex, alpha, beta, and subunits covalently attach to the primer-template in an ATP-dependent reaction and that a change in subunit-DNA contacts occurs during the transition from a preinitiation to an initiation complex.


MATERIALS AND METHODS

Proteins and Enzymes

E. coli DNA Pol III holoenzyme (^2)(700,000 units/mg) was prepared by the method of Oberfelder and McHenry(1987), DNA Pol III` (^3)(400,000 units/mg) by the method of McHenry(1982), and core DNA Pol III^3 (850,000 units/mg) by the method of McHenry and Crow(1979). The -complex (18.7 times 10^6 units/mg) was provided by Dr. A. J. Hughes, Jr. of this laboratory and contains the , , `, , and subunits. beta subunit (10.0 times 10^6 units/mg) was isolated by standard procedure (Johanson and McHenry, 1980). These proteins are also available from Enzyco, Inc., Denver, CO. SSB (0.7 mg/ml) was isolated from an E. coli overproducer, RLM727 (constructed by and gift of Roger McMacken) and purified by a modification (Griep and McHenry, 1989) of the method described by Meyer et al. (1980). Sequenase version 2.0 T7 DNA polymerase was purchased from United States Biochemical Corp. (Cleveland, OH).

Nucleotides and Nucleic Acids

Unlabeled ATP was obtained from Pharmacia and [alpha-P]ddATP (>5000 Ci/mmol) was purchased from Amersham Corp. M13mp19 single-stranded DNA template was isolated by the procedure of Johanson and McHenry(1984) and provided by Mary Ann Franden of this laboratory.

Reagent Preparation

Dichloromethane (Aldrich) was dried as described by Perrin and Armarego(1988), refluxed over CaH(2) (Fisher) for 2 h, and the distillate was collected at 35 °C. 4,4`-Dimethoxytrityl chloride was dried overnight in an abderhalden drying apparatus containing P(2)O(5) (Aldrich).

Synthesis of 5-(3-Aminopropyl)-2`-deoxyuridine phosphoramidite

Freshly distilled N-propargyltrifluoroacetamide, prepared from propargylamine (Aldrich) and trifluoroacetic anydride (Aldrich), was coupled in good yield with 5-iodo-2`-deoxyuridine (Fluka) according to the method of Robins et al.(1990). The resulting crude product was purified by flash chromatography on silica gel utilizing a step gradient (1 column volume each of 75/25, 50/50, and 75/25 methylene chloride/ethyl acetate). The structure of the product was confirmed by proton NMR (200 MHz). The propynyl intermediate (3.6 g) was dissolved in 60 ml of absolute ethanol containing 20 ml of ethyl acetate. Following the addition of 300 mg of hydrogenation catalyst (5% Pd on C), the reaction was placed on a Parr hydrogenation apparatus and was complete in 2 h. The catalyst and solvent were removed, and the product was crystallized from absolute ethanol (2.6 g, 86%). Proton NMR revealed that the reduction had proceeded as desired. The 5` OH of the reduced nucleoside was dimethoxytritylated by treatment with demethoxytrityl chloride (Aldrich) as outlined by Jones(1984) and purified by silica gel chromatography (2.5 times 18 cm; 0-10% methanol in methylene chloride). The 3`-phosphoramidite derivative was prepared from the 5` blocked compound according to the methods described (Barone et al., 1984; Sinha, 1984; Atkinson and Smith, 1984). The resulting 3`-phosphoramidite was precipitated upon the dropwise addition of an ethyl acetate solution of the product to a precooled (-80 °C) solution of reagent grade hexane. The dried, precipitated 3`-phosphoramidite compound was sufficiently pure (thin layer chromatography) for direct incorporation into oligonucleotides.

Synthesis and Purification of the Oligonucleotide

Two 24-mers, four 30-mers, and one 50-mer (complementary to M13mp19 at nucleotides 991-1015, 991-1021, and 991-1041, respectively) (Yanisch-Perron et al., 1985) were synthesized on a BioSearch 8600 DNA synthesizer. A cytidine-controlled pore glass support (1 µmol) (MilliGen/Biosearch) was used in order to position a ribonucleotide at the 3` terminus of each synthesized deoxynucleotide primer (Fig. 1A). The terminal 5`-dimethoxytrityl protecting group was left on the oligomers to facilitate purification by reversed phase HPLC. The trifluoroacetate-protected 5-(3-trifluoroacetamide-1-propyl)-2`-deoxyuridine was incorporated into each of the seven oligomers at a unique site. Six of the oligomers had a modified residue positioned within a 30-nucleotide region to accommodate the occupancy site of holoenzyme as defined by DNase I footprint analysis (Reems and McHenry, 1994) at positions 2, 8, 12, 17, 21, and 26 upstream from the 3`-primer terminus; the seventh oligomer had the modified residue positioned 45 nucleotides upstream of the 3`-primer terminus (Fig. 2). Cleavage from the controlled pore glass support and removal of all protecting groups except the 5`-dimethoxytrityl group were carried out as described (Hagerman, 1985). The trityl-protected oligonucleotides were purified by HPLC on a Waters µBondapak C(18) column (3.9 times 300 mm) using a gradient of acetonitrile in 90 mM triethylammonium acetate, pH 7.0 (flow rate, 0.8 ml/min; Program: 0-4 min, 0% acetonitrile wash; 4-54 min, concave gradient to 50% acetonitrile; 54-59 min, concave gradient to 100% acetonitrile; 59-68 min wash). Continuous monitoring at 260 nm (Waters 994 photodiode array detector) indicated that the trityl-protected oligonucleotides eluted at 58 min for the 24-mers, 46 min for the 30-mers, and 51 min for the 50-mer. The tritylated oligomers were deprotected with 6% acetic acid, 10 min at room temperature, and purified by HPLC using the same conditions as for the tritylated oligomers (Fig. 1B). Retention time after detritylation for each of the oligomers was 47 min. Stock primer concentrations were determined using extinction coefficients derived by the summation of the individual base extinction coefficients (Borer, 1975). Average yield from a 1-µmol oligonucleotide synthesis for purified detritylated oligomers was between 5-10%. The oligomers were evaporated to dryness in a Speed-Vac (Savant Instruments).


Figure 1: Chemical steps in the preparation of phenylazide photo-cross-linking primers. A, oligonucleotide with trifluoroacetate-protected propylamine deoxyuridine at position -2. B, oligonucleotide after removal of the trifluoroacetate-protecting group. C, primer after derivatization with N-hydroxysuccimidyl-5-azido-nitrobenzoate. D, primer after 3` extension with [P]ddATP, placing the photo-reactive probe at position -3.




Figure 2: Nucleotide sequence and position of the photo-reactive probe for each of the seven derivatized primers. Seven different primers containing uniquely positioned photo-reactive aryl azides were annealed to a derivative of M13 and labeled at the primer 3`-terminus with [alphaP]ddATP using deoxynucleotidyl transferase as described under ``Materials and Methods.'' Each of the seven derivatized primer-templates possesses a 3`-terminal dideoxyadenosine, a 3`-penultimate ribonucleotide (cytidine), and a single photo-reactive probe. The final probe position after annealing the derivatized primer to M13 and radiolabeling the 3` terminus is indicated to the left of each primer. The photo-reactive probe is positioned either 3, 9, 13, 18, 22, 27, or 46 (-3, -9, -13, -18, -22, -27, and -46, respectively) nucleotides upstream from the primer 3` terminus. U* = propylamine-deoxyuridine derivatized with phenylnitroazide.



Derivatization of Primers with N-Hydroxy-succinimidyl-5-azido-2- nitrobenzoate

The detritylated oligonucleotides were derivatized with the cross-linking reagent N-hydroxy-succinimidyl-5-azido-2-nitrobenzoate (Pierce) as described by Gibson and Benkovic(1987) with the following modification. The dried detritylated oligomers were dissolved in 100 mM sodium carbonate, pH 9.5, prior to treatment with the cross-linking reagent. Derivatized oligomers were purified on the Waters C18 reverse-phase column using the same conditions and gradient as described above and eluted at 47 min. Peak fractions containing photo-reactive oligomers exhibited UV spectra (Waters 994 photodiode array detector) maximum absorption at 258 nm and a shoulder peak at 320 nm, characteristic of the ring system containing the azido group.

Exonuclease Assay

Two 24-mers, the first with the photo-reactive aryl azide positioned at -2 and the second without the photo-reactive group, were radiolabeled at the 5` terminus using T4 polynucleotide kinase and [-P]ATP according to described procedures (Maniatis et al., 1982). A premix containing 50 mM HEPES, pH 7.5, 100 mM potassium glutamate, 10 mM dithiothreitol, 500 µM ATP, 10 mM magnesium acetate, 2.0 nM primed template, and 2.0 µg SSB/nmol nucleotide was incubated for at least 2 min at 30 °C. Three aliquots (12.5 µl) were removed prior to the addition of 3-4 units of holoenzyme/fmol of primer-template. After holoenzyme addition, 12.5-µl aliquots were withdrawn at various time points and quenched with a final concentration of 140 mM EDTA. Quenched reaction mixtures were transferred onto Whatman DE-81 filters and batch-washed (three times, 5 min each in 0.3 M ammonium formate, pH 7.8, 10 mM sodium pyrophosphate; once in deionized water; and once in 95% ethanol). Filters were dried, and the amount of radiolabel remaining was measured by scintillation counting. Infinity points were determined by adding 3-4 units of holoenzyme/fmol primer-template and allowing the reaction to proceed for 10 half-lives. An additional amount of holoenzyme (3-4 units/fmol primer-template) was added, and the reaction was allowed to proceed for another 10 half-lives. Three aliquots (12.5 µl) were removed and the radiolabel quantitated as described above. Data were analyzed as described (Griep et al., 1990).

Elongation Assay

The elongation of a primer-template by holoenzyme was determined by using a modification of previously described procedures (Johanson and McHenry, 1984; Griep and McHenry, 1989). Briefly, the final reaction mixture contained either 2.0 nM primer-template in 50 mM HEPES, pH 7.5, 500 µM ATP, 10 mM magnesium acetate, 0.01% (v/v) Nonidet P-40, 80 µg/ml bovine serum albumin, or 1.0 nM primer-template, 2.0 µg SSB/nmol nucleotide, and 500 µM each of dATP, dCTP, dGTP, and dTTP. Each reaction mixture was equilibrated to 30 °C for at least 2 min prior to the addition of 3-4 units of holoenzyme/fmol primer-template. After a 5-min incubation, the reaction was quenched with 90% formamide and loaded onto an 8% polyacrylamide, 8 M urea sequencing gel.

Exonuclease-resistant Photoreactive Primer-Templates

The presence of a terminal dideoxyadenosine coupled to a penultimate ribonucleotide reduces the 3`5` exonuclease activity of holoenzyme nearly 1000-fold (Griep et al., 1990). To generate radioactive exonuclease-resistant primers, each of the photo-reactive oligonucleotides containing a ribonucleotide positioned at the 3` terminus was extended with [alpha-P]ddATP after hybridizing each oligomer to M13mp19. This was accomplished by mixing oligomer (800 nM) with M13mp19 (400 nM) in 50 mM HEPES (pH 7.5), 200 mM NaCl at 75 °C for 5 min followed by slow cooling (1.5 °C/min) to room temperature. Primer-template (200 nM) was incubated with [alpha-P]ddATP (1.0 µM), 3 units of Sequenase/pmol of primer-template in 10 mM magnesium acetate, 50 mM HEPES (pH 7.5) for 30 min at 37 °C. Enzyme was thermally inactivated (10 min, 65 °C). The labeled primer-template was separated from free primer and unincorporated nucleotide on a Bio Gel A-5 m column (1.0 times 3.0 cm) (Bio-Rad) equilibrated in 50 mM HEPES (pH 7.5), 100 mM potassium glutamate, 1 mM EDTA. Primer-template concentrations were determined spectrophotometrically at 260 nm and were presented in terms of primer 3`-hydroxyl termini. Seven oligonucleotides containing aryl azide-substituted U at position 3, 9, 13, 18, 22, 27, or 46 upstream of the 3`-ddA terminus were prepared (Fig. 2).

Site-specific Photo-cross-linking of Enzyme to Primer-Template

Saturating levels of enzyme (see figure legends) were added to 50 mM HEPES (pH 7.5), 500 µM ATP, 10 mM magnesium acetate, 0.01% (v/v) Nonidet P-40, 80 µg/ml bovine serum albumin, 4.0 nM photo-reactive primer-template, and 2.0 µg SSB/nmol nucleotide (25 µl). EnzymebulletDNA complexes were incubated at 30 °C for 1 min prior to photo-irradiation. Photolysis was accomplished using an SLM-Aminco 48000 spectrofluorometer. The samples were photolyzed at a wavelength of 315 nm (450 W xenon arc lamp, 16 nm bandpass) at a distance of 145 cm for 10 min (15 milliwatts/cm^2). An equal volume of sample buffer (50% glycerol, 10 mM dithiothreitol, 0.2% bromphenol blue, 0.1 M Tris, pH 6.8, 1.0% SDS) was added to each reaction prior to loading the samples onto a 5-20% SDS-polyacrylamide gel. After electrophoresis, gels were silver-stained, transferred to Whatman 3MM paper, and dried. To visualize the resolved cross-linked products, gels were exposed to Kodak film (SB or X-OMAT AR) at room temperature or exposed to a PhosphoroImager screen (Molecular Dynamics). Cross-linked subunits were identified by their molecular weights and gel mobilities relative to a holoenzyme standard. Molecular weights and R(f) values were calculated for each radioactive photo-product and for each subunit present in the holoenzyme standard.

Site-specific Photo-cross-linking of Enzyme to Unannealed Primer

A 24-mer with the reporter group positioned 9 nucleotides upstream of the 3`-primer terminus was 5`-phosphorylated using T4 polynucleotide kinase and [-P]ATP according to a procedure by Maniatis et al.(1982). Radiolabeled oligonucleotide was gel-filtered and quantitated as described above. Saturating levels of enzyme (see figure legends) were incubated with unannealed primer (4.0 nM) in 50 mM HEPES (pH 7.5), 500 µM ATP, 10 mM magnesium acetate, 0.01% (v/v) Nonidet P-40, 80 µg/ml bovine serum albumin, and 2.0 µg SSB/nmol nucleotide (25 µl). After 1 min at 30 °C, samples were photo-irradiated, and the cross-linked products were identified as described for enzyme-primer-template complexes.


RESULTS

In the presence of ATP, the multi-subunit E. coli DNA Pol III holoenzyme (alpha, , beta, , , , `, , , and subunits) forms a highly stable initiation complex with a primed template (Wickner and Kornberg, 1973; Wickner, 1976; Burgers and Kornberg, 1982a, 1982b; Johanson and McHenry, 1980, 1982). To identify the subunit-DNA interactions that occur within the initiation complex, we mapped the linear arrangement of the holoenzyme subunits along the DNA helix using site-specific photo-cross-linking.

Our previous work from DNase I footprint studies indicated that holoenzyme protects 30 nucleotides of primer (Reems and McHenry, 1994). Based on the footprint data, we synthesized seven different primers with a single photo-reactive aryl azide group uniquely placed within the holoenzyme binding region (Fig. 2), adapting the synthetic methods originally developed by Gibson and Benkovic(1987) to synthesize primers containing uniquely positioned photo-affinity labels. This method involved the introduction of a trifluoroacetate-protected propylamine to deoxyuridine, synthesis of protected phosphoramidite, and insertion of the trifluoroacetate-protected propylamine deoxyuridine phosphoramidite into an oligonucleotide chain. Standard deblocking procedures removed the trifluoroacetate protecting group from the propylamine which was then derivatized with the photo-reactive agent N-hydroxy-succinimidyl-5-azido-nitrobenzoate in preparation for the photo-cross-linking studies (Fig. 1). Each of the primers was complementary to single-stranded circular bacteriophage M13mp19.

To map the linear arrangement of the holoenzyme subunits along the DNA helix, we photo-irradiated holoenzyme-primer-template complexes bearing a photo-reactive group within the primer strand. Seven different derivatized primers were separately annealed to M13mp19, and the 3` terminus of each primer was radiolabeled. The photo-reactive probe position for each of the seven primers was 3, 9, 13, 18, 22, 27, or 46 nucleotides upstream from the primer 3` terminus (Fig. 2). By changing the position of the photo-reactive probe relative to the 3` terminus of the primer, photolysis of holoenzyme primer-template complexes permitted the identification of different subunit-DNA contacts along the primer-template in a linear fashion.

Photo-products were resolved on a 5-20% SDS-polyacrylamide gel with no stacking gel. Each of the gels showed two radioactive bands corresponding to primer-template that did not enter the gel (top of gel) or to cross-linked SSB (bottom of gel) (Fig. 3). Holoenzyme subunits that were cross-linked to the primer were determined by the gel mobility of the cross-linked species relative to free protein. Cross-linked photo-products exhibited gel mobilities slightly slower than their non-cross-linked counterparts due to the molecular weight of the covalently attached primer. In addition to non-cross-linked molecular weight markers, the individually cross-linked proteins alpha and SSB were used as markers.


Figure 3: Subunit-DNA adducts formed after cross-linking E. coli DNA polymerase III holoenzyme to six different photo-reactive primer-templates. Reaction conditions were as described under ``Materials and Methods.'' Initiation complexes were formed using 3 units of holoenzyme/fmol of primer-template and 1.0 mM ATP prior to photolysis. A control reaction for each probe position was performed by photo-irradiating each primer-template in the absence of holoenzyme. The position of the photo-reactive group relative to the 3` terminus of the primer is indicated above each pair of lanes.



Primer-Templates Containing a Photo-reactive Aryl Azide Group Are Effective Substrates for Holoenzyme

To determine whether primers containing a photo-reactive aryl azide were suitable substrates for holoenzyme, we monitored enzyme function using derivatized primer-templates. A derivatized primer bearing a photo-reactive group at position -2 and a radioactive label at the 5` terminus was annealed to M13mp19 and incubated with holoenzyme in the presence of all four dNTPs. Primer extension was monitored using a 6% sequencing gel. In the presence of all four dNTPs, holoenzyme elongated both derivatized and non-derivatized primers to full-length product with no detectable intermediate fragments (data not shown).

To monitor the 3`5` exonuclease activity of holoenzyme, we compared the enzyme's excisional activities using primers with or without a photo-reactive group. When holoenzyme was incubated with primer-template in the absence of all four dNTPs, the enzyme fully degraded derivatized primers in 90 s and non-derivatized primers in 45 s. Even though there was a 2-fold reduction in exonuclease activity using derivatized primers (data not shown), the primer was fully degraded within the time frame of our photo-cross-linking experiments. Thus, the aryl azide did not significantly interfere with holoenzyme's synthetic or proofreading activities. However, to overcome the technical difficulties for our experiments presented by holoenzyme's ability to rapidly degrade DNA primers, we made two alterations in the primer to reduce its susceptibility to the enzyme's 3`5` exonuclease activity. First, we rendered the primer exonuclease-resistant by positioning a penultimate ribonucleotide and a terminal dideoxyribonucleotide residue at the 3` terminus. These modifications reduced the exonuclease activity to stabilize holoenzyme-DNA primer-template interactions nearly 1000-fold (Griep et al., 1990). Second, we radiolabeled the 3` rather than the 5` terminus of the primer, ensuring that the 3`5` exonuclease activity would cleave the radiolabel in the form of a mononucleotide, thus eliminating ambiguity in cross-link assignments since only photo-products that occurred prior to excision would be detectable. These alterations made it possible for us to detect photo-reactive products due only to the formation of static holoenzyme-primer-template complexes, which is necessary in determining the linear alignment of holoenzyme subunits relative to the primer-template.

E. coli DNA Polymerase III Holoenzyme Subunits, alpha, beta, and Directly Contact the Primer-Template

Photolysis of holoenzyme incubated with derivatized primer-template containing an aryl azide at the -3 position resulted in a radioactive band that migrated to the position expected for cross-linked alpha (Fig. 3) and required the presence of ATP. (^4)

We then cross-linked holoenzyme to the remaining derivatized primers (Fig. 3). Primer-template with the aryl azide positioned at -9 resulted in one highly radioactive band that corresponded to cross-linked alpha. Photo-irradiation of derivatized primer-template, aryl azide positioned at -13, resulted in faint photo-products that corresponded to the gel positions expected for cross-linked alpha and . Holoenzyme-primer-template complexes photo-irradiated with the aryl azide positioned at -18 resulted in only one highly radioactive photo-product whose gel migration corresponded to cross-linked . At position -22, the photo-products migrated to positions expected for cross-linked and beta, and at position -27, there were no apparent photo-products. Cross-linking at position -46 was also not detected (data not shown).

SSB photo-cross-linked to all of the annealed primers used in these experiments. All complexes were isolated by gel filtration to eliminate free primers, but we cannot absolutely rule out the possibility that trace quantities dissociated after isolation. Presumably, SSB can interact with the duplex region, albeit weakly. Previously, we observed that SSB did not protect annealed primers from DNase I digestion. When interpreting these results it should be kept in mind that one cannot compare the cross-linking intensity for two separate proteins to estimate their binding strength. Cross-linking efficiency varies markedly between proteins and is determined by the proximity of reactive amino acids to the photoreactive group. Capson and co-workers (et al., 1991) made similar observations with the T4 gene 32 protein cross-linking to annealed photoreactive primers.

Several subassembly forms of DNA polymerase III are known to interact with primer-template in an ATP-independent mode. However, maximum synthetic and 3`5` exonuclease activities for holoenzyme are achieved only when ATP is present. Analysis of ATP requirements to confirm that the cross-linked products were due to holoenzyme and not subassembly forms revealed that cross-linking of the alpha, beta, and subunits to primer required ATP (data not shown).

The , (or `), and beta Subunits of the Preinitiation Complex Directly Cross-link to Primer in an ATP-dependent Reaction

To identify subunit contacts made with the primer during preinitiation complex formation, we photo-cross-linked the components of the preinitiation complex (beta plus -complex (, , `, , and )) to a derivatized primer-template. Since exonuclease activity is associated with the subunit which is not part of the preinitiation complex, primers with a radiolabel at the 5` terminus instead of the 3` terminus were used. The aryl azide was positioned 2 nucleotides upstream of the 3` terminus.

Photo-cross-linking the components of the preinitiation complex to primer-template in the presence of both ATP and SSB resulted in the covalent attachment of the , beta, and (or `) subunits (Fig. 4A, lane 1; in the system used, the and ` subunits cannot be distinguished). By eliminating ATP from the reaction, both the and beta subunit interactions with the primer-template were precluded (Fig. 4A, compare lanes 1 and 5), whereas (or `) primer-template interactions remained essentially unchanged (Fig. 4A, compare lanes 1 and 5). The radioactive band, designated times, between the beta and subunits was apparent even in the absence of any enzyme (Fig. 4A, lanes 4 and 8), indicating that this photo-product was not due to cross-linked holoenzyme subunits.


Figure 4: Subunit-DNA adducts formed after photo-cross-linking components of the preinitiation complex (beta plus -complex subunits) to primer-template in the presence or absence of SSB. Reaction conditions were described under ``Materials and Methods.'' The primer was radiolabeled at the 5` terminus. The photo-reactive probe was positioned 2 nucleotides upstream of the 3` terminus. Enzyme-primer-template complexes were formed with 10 units of -complex, and/or 70 units of beta subunit/fmol of primer-template in the presence or absence of 500 µM ATP. A, photo-products obtained after irradiating enzyme-primer-template in the presence of 2.0 µg SSB/nmol of nucleotide. B, photo-products obtained after irradiating enzyme-primer-template in the absence of SSB.



Photo-cross-linking of the -complex in the absence of the beta subunit still resulted in and (or `) interactions with the primer. In the presence of ATP, within the -complex cross-linked to the primer and did not require the addition of the beta subunit (Fig. 4A, lane 2). In the absence of ATP, interactions with the primer were almost eliminated (Fig. 4A, lane 6). Photo-cross-linking of the beta subunit without the -complex did not result in beta-DNA adduct formation (Fig. 4A, lane 3). Further, no apparent beta-DNA adducts were obtained even when using excess beta and a primer containing an aryl azide positioned at -22 (data not shown). Thus, beta interactions with primer required both -complex and ATP (Fig. 4A, compare lane 1 to lanes 4 and 7), results which are consistent with the proposal that the -complex transfers the beta subunit to primer-template in an ATP-dependent reaction (Wickner 1976; O'Donnell, 1987).

SSB Interferes with (or `) ATP-independent Binding

SSB has several known functions during DNA replication, including its ability to enhance polymerase processivity and to promote polymerase binding to primer-template (for review, see Meyer and Laine, 1990). To investigate the role of SSB in the binding of holoenzyme to primer-template, we cross-linked preinitiation complexes to primer-template in the presence or absence of SSB (Fig. 4). Photo-cross-linking components of the preinitiation complex to primer-template in the presence of ATP without SSB resulted in the covalent attachment of the , beta, and subunits (Fig. 4B, lane 9). However, beta cross-linking was more prevalent than in the absence of SSB, while formation of and beta-adducts to the primer was equivalent in the presence of SSB. By eliminating ATP from the reaction, both and beta interactions with the primer-template were precluded (Fig. 4B, lane 13).

Surprisingly (or `)-primer interactions were dramatically different in the absence or presence of SSB. In the absence of SSB, the amount of (or `) that cross-linked to the primer was increased 5-10-fold (compare Fig. 4, A to B). Further, (or `) interactions with the primer-template were essentially the same with or without ATP (Fig. 4B, compare lanes 9 and 10 to lanes 13 and 14). These results indicate that SSB competes with the (or `) subunit for binding to the primer-template at a position 2 nucleotides upstream of the 3` terminus of the primer. Further, (or `) interactions with the primer-template occur in an ATP-independent fashion.

Holoenzyme Undergoes a Conformational Change during Initiation Complex Formation

To identify subunit-DNA contacts that occur during the formation of holoenzyme-primer-template complexes, we photo-cross-linked partially reconstituted complexes and analyzed intermediate subunit-DNA interactions. Photo-irradiation of primer-template incubated with either core Pol III or Pol III` resulted in 10-fold less alpha-DNA adducts than with holoenzyme-primer-template complexes (Fig. 5, compare lanes 2 and 3 to lane 7). Within the preinitiation complex, the , beta, and subunits cross-linked to the primer 2 nucleotides upstream from the 3` terminus (Fig. 5, lane 4). However, with the addition of core Pol III or Pol III` to the preinitiation complex, the , beta, and (or `) subunits were replaced by the alpha subunit (Fig. 5, lanes 5 and 6). These results suggest that a conformational change occurs upon the binding of either core Pol III or Pol III` to preinitiation complexes.


Figure 5: Photo-products generated using individual components of DNA polymerase III holoenzyme initiation complexes. Reaction conditions were as described under ``Materials and Methods.'' The primer was radiolabeled at the 5` terminus. The photo-reactive probe was located 2 nucleotides upstream of the 3` terminus. Enzyme-primer-template complexes were formed using 3 units of holoenzyme, 10 units of -complex, 70 units of beta, 3 units of core Pol III, and/or 3 units of Pol III`/fmol of primer-template in the presence of 500 µM ATP. These additions resulted in enzyme/primer molar ratios of 6:1, 18:1, and 20:1 for holoenzyme, core pol III and pol III`, respectively. beta and complex, when included, were present at enzyme/primer ratios of 18:1 and 86:1, respectively. Enzyme additions are indicated above each lane.



Holoenzyme Subunits alpha, beta, and Cross-link to Primer-Template and Not to Unannealed Primer

To ensure that cross-linked photo-products directly reflect holoenzyme interactions with primer-template and not with unannealed primer, we examined holoenzyme's ability to interact with unannealed primer using two approaches. The first approach involved incubating holoenzyme or -complexes with derivatized unannealed primers and then photo-irradiating the enzyme-primer complexes; no apparent cross-linking of the alpha subunit occurred with unannealed primer when using holoenzyme (Fig. 6, compare lanes 3-6), and no apparent cross-linking of the , , (or `) or beta subunits occurred to unannealed primer when using the -complex (Fig. 6, compare lane 2 to 5). The second approach was a competition assay in which a nonspecific competitor, i.e. unannealed primer-template or M13mp19 alone, or a specific competitor, i.e. non-derivatized primer annealed to M13, was added before holoenzyme to the primer-template. A 10-fold molar excess of specific competitor successfully competed alpha cross-linking to a primer with the photo-reactive group at position -3 (Fig. 7, lane 1), whereas a 100-fold molar excess of nonspecific competitor failed to compete alpha cross-linking to the primer (Fig. 7, lanes 2 and 3). Thus, the subunit-DNA photo-products were detected only when enzyme interacted with primer annealed to a template strand.


Figure 6: Subunit-DNA adducts occur with primer-template and not with primer alone. Reaction conditions were as described under ``Materials and Methods.'' The primer was radiolabeled at the 5` terminus. The position of the photo-reactive probe was 9 nucleotides upstream of the 3` terminus. Enzyme-primer-template complexes were formed using 3 units of holoenzyme, 10 units of -complex, and 70 units of beta subunit/fmol of primer-template in the presence of 500 µM ATP. Photo-products obtained after irradiating enzyme that had been incubated with either primer alone or primer-template.




Figure 7: Excess unlabeled primer-template competitively inhibits subunit-DNA adducts with radioactive primer-template. Reaction conditions were as described under ``Materials and Methods.'' Lane 1, excess unlabeled primer-template (P:T) (100 nM), was incubated with radioactive photo-reactive primer-template (4.0 nM) 50 mM HEPES, pH 7.5, 500 µM ATP, 10 mM magnesium acetate, 0.01% (v/v) Nonidet P-40, 80 µg/ml bovine serum albumin, and 2.0 µg SSB/nmol nucleotide (25 µl) for 5 min at 30 °C prior to the addition of subsaturating levels of holoenzyme (0.5 unit/fmol primer-template), incubated for 1 min at 30 °C, and photo-irradiated. Lanes 2 and 3, excess unlabeled single-stranded circular template strands (500 nM) or unannealed primer (500 nM) were added to labeled photo-reactive primer-template (4.0 nM). After 5 min at 30 °C, holoenzyme (0.5 unit/fmol primer-template) was added to the reaction, incubated for 1 min at 30 °C and photo-irradiated. Lane 4, positive control where no competitor was added before holoenzyme-primer-template complexes were formed. Lane 5, negative control where no competitor holoenzyme were added.




DISCUSSION

The purpose of this study was to map the linear arrangement of the holoenzyme subunits along the duplex region of the primer-template at the initiation site. To accomplish this goal, we used photo-cross-linking to site-specifically identify holoenzyme subunit contacts relative to a DNA primer. These studies revealed that the alpha subunit cross-linked to positions -3, -9, and -13, with the most efficient cross-linking occurring at position -9. The subunit cross-linked to positions -13, -18, and -22, with the strongest -primer interactions occurring at position -18. The beta subunit was the predominant protein cross-linked at position -22, while at positions -27 and -45, essentially no holoenzyme contacts were noted. Together these results indicate that within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3`-primer terminus, followed by at -18 and beta at -22 (Fig. 8).


Figure 8: The linear alignment of E. coli DNA polymerase III holoenzyme subunits relative to the duplex region of the primer-template at the initiation site. Schematic representation of the holoenzyme subunit-DNA contacts defined by site-specific photo-cross-linking. The alpha subunit, possessing the polymerase catalytic site, covalently attaches to positions -3, -9, and -13, with the most efficient cross-linking occurring at position -9. The subunit cross-links to positions -13, -18, and -22, with the strongest -primer interactions occurring at position -18. The beta subunit is the predominant subunit covalently attached to the primer at position -22. Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3`-primer terminus followed by at -18 and beta at -22. remains part of the initiation complex connecting the alpha and beta subunits.



We previously reported the use of fluorescence energy transfer to map the position of the beta subunit within the initiation complex (Griep and McHenry, 1992). In that study, a donor-acceptor distance of 65 Å was determined between a donor fluorophore located at the -3 position of the primer and an acceptor fluorophore positioned within the beta subunit at Cys, a distance which positions the beta subunit approximately 19 nucleotides from the donor fluorophore or 22 nucleotides upstream of the 3`-primer terminus. This distance is consistent with the present results which show that beta covalently attaches to the primer 22 nucleotides upstream of the 3`-primer terminus. DNase I footprint analysis (Reems and McHenry, 1994) of holoenzyme binding to the primer-template showed that holoenzyme contacts 30 nucleotides of primer-template; photo-cross-linking results indicate that holoenzyme contacts at least 22 nucleotides of primer. Since DNase I cuts occur at 4-nucleotide intervals and would be expected to be sterically hindered from cutting immediately adjacent to a protein on DNA, it is reasonable to find a slightly larger region defined by DNase I footprint analysis than the site-size defined by photo-cross-linking.

Interestingly, the T4 DNA polymerase genes 43, 44/62, and 45, which are analogous to the E. coli DNA polymerase III alpha, -complex, and beta subunits, respectively, map to similar positions. Gene 43, which is responsible for nucleotide incorporation in the T4 DNA polymerase replication complex, contacts position -4 upstream of the 3`-primer terminus. Gene 62, which is part of the ATP accessory complex, contacts the primer at position -9, and gene 45, the T4 processivity factor, contacts the primer at positions -14 and -20 (Capson et al., 1991). The similar structural position and order of the T4 replication machine components is consistent with their functional equivalences, predicting that this order might be conserved among all replication machines. To extend this prediction to eukaryotic replication enzymes, one might expect polymerase to interact with the primer terminus, a component of 5-protein RFC to interact at an adjacent position, and the PCNA sliding clamp to reside at the most distal position in eukaryotic replication complexes. Of course, extra interactions may be present to accommodate other interactions including the polymerase-primase complex.

DNase I footprint analysis and fluorescence energy transfer studies also suggested that subunit rearrangements occur during formation of holoenzyme-primer-template complexes (Griep and McHenry, 1992; Reems and McHenry, 1994). We, therefore, photo-cross-linked partially reconstituted complexes in order to isolate and analyze intermediate subunit-DNA interactions. Photo-cross-linking preinitiation complexes to primer-template indicated that , beta, and subunits contact the primer at position -2. However, with the addition of core Pol III or Pol III` to the preinitiation complex, the alpha subunit replaces , beta, and subunits at position -2. These results provide direct evidence that the binding of core polymerase to preinitiation complexes induces a subunit rearrangement.

Our map of the linear alignment of the holoenzyme subunits along the DNA helix provides structural information central to defining the functional activities of the polymerase subunits within the initiation complex. Our data indicating that alpha contacts at least 13 nucleotides of the primer upstream of the 3`-primer terminus positions this subunit, which possesses the catalytic site for the polymerization reaction, at the primer-template junction poised to incorporate dNTPs at the 3`hydroxyl terminus of the primer. At positions -18 and -22, respectively, and beta contact the primer. The beta subunit, which confers processivity to the core polymerase (Fay et al., 1981) and functions as a ``sliding clamp'' (Stukenberg et al., 1991), is situated upstream of the alpha and subunits, placing the processivity factor approximately two helical turns upstream from the 3`-primer-terminus assuming a beta-form DNA conformation. The -complex recognizes the primer-template and couples ATP hydrolysis to clamp beta at the initiation site (Onrust et al., 1991). The positioning of between alpha and beta suggests a role for in locking and linking the processivity and elongation factors. Several laboratories have reported that the complex functions to load beta(2) onto the primer-template and then dissociates. Our results suggest a participatory role for the complex in elongation.


FOOTNOTES

*
This work was supported by Research Grant RO1 GM 35695 from the National Institutes of General Medical Sciences and facilities support from the Lucille Markey Charitable Trust. 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.

§
Present address: Fred Hutchinson Cancer Research Center, M318, Seattle WA 98104.

Present address: Dept. of Botany and Range Science, 401 WIDB, Brigham Young University, Provo, UT 84602.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: holoenzyme, E. coli DNA polymerase III holoenzyme (alpha, , beta, , , , `, , , and subunits); SSB, E. coli single-stranded DNA-binding protein; Pol III*, E. coli DNA polymerase III* (holoenzyme minus the beta subunit); Pol III`, E. coli DNA polymerase III` (alpha, , , and subunits); core Pol III, E. coli DNA polymerase III core (alpha, , and subunits); -complex, , , `, , and subunits; preinitiation complex, beta plus -complex; HPLC, high performance liquid chromatography.

(^2)
One unit of holoenzyme activity is defined as 1 pmol of (total) deoxynucleotide incorporated/min on a G4 DNA template with priming by dnaG primase in situ.

(^3)
One unit of Pol III or Pol III` activity is the amount of enzyme catalyzing the incorporation of 1 pmol of (total) deoxynucleotide/min on an activated salmon sperm DNA template.

(^4)
In lane(-3+), Fig. 3, we observed a highly radioactive band migrating in the position expected from . This band was highly variable in most of the experiments reported in this paper and was further confused by appearance even when holoenzyme was deleted (see lane 22(-), Fig. 3) The extra band could be due to DNA-DNA cross-links or a related artifact. Thus, we cannot make any definitive statement about the appearance of .


ACKNOWLEDGEMENTS

We thank Mark Seville, John Hughes, and Doug Dellinger for helpful discussions. We are especially grateful to Steve Benkovic and Katherine Gibson for their advice and for supplying derivatized primers for our preliminary studies.


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