THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 5, Issue of March 10. pp. 2657-2663, 1982 Printed in U.S.A.

Purification and Characterization of DNA Polymerase 111’ IDENTIFICATION OF 7 AS A SUBUNIT OF THE DNA POLYMERASE I11 HOLOENZYME*

(Received for publication, September 24, 1981)

Charles S. McHenry From the Department of Biochemistry and Molecular Biology, The University of Texas Medical School, Houston, Texas 77025

DNA polymerase 111’, a new form of DNA polymerase 111, has been purified 15,000-fold to 90% homogeneity from an Eschericlria coli K12 strain. DNA polymerase III’ is a subassembly of four subunits of the DNA polym- erase 111 holoenzyme; it has functional and physical properties intermediate between the core DNA polym- erase III and holoenzyme. Polyacrylamide gel electro- phoresis performed under denaturing conditions indi- cates DNA polymerase 111’ to be a complex of the a, E , and B subunits of DNA polymerase 111 and a newly assigned subunit of the DNA polymerase 111 holoen- zyme, T (Mr = 83,000). Both gel filtration and phospho- cellulose chromatography separate DNA polymerase 111 from DNA polymerase III’. AU enzyme forms can utilize a duplex template containing short gaps. DNA polymerase 111’, like the DNA polymerase 111 holoen- zyme, can synthesize DNA on a long single-stranded template in the presence of 5 m~ spermidine; DNA polymerase 111 cannot. Alone, DNA polymerase 111’ is inert in the G4 natural replicative system in which the DNA polymerase 111 holoenzyme is active. Molecular weight and subunit stoichiometry determinations sug- gest that DNA polymerase III’ contains two units of core DNA polymerase 111 and two T subunits.

The DNA polymerase I11 holoenzyme’ is a complex multi- subunit enzyme which has been implicated by biochemical and genetic studies as the polymerase responsible for the majority of replicative DNA synthesis in E. coli (for reviews, see Kornberg, 1980; McHenry and Kornberg, 1981). Although a final determination of the DNA polymerase I11 holoenzyme structure has not been accomplished, at least three auxiliary proteins, p, y, and 8, are required in addition to the core DNA polymerase I11 for conversion of primed G4 and @X 174 single strands to the duplex replicative form (McHenry and Korn- berg, 1977). Wickner and Hurwitz (1976) have isolated three proteins, Factor I, dna Z protein, and Factor 111, which may correspond to p, y , and 8, respectively (McHenry and Korn- berg, 1977).

* This work was supported by a grant from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

DNA polymerase I11 holoenzyme is the term used for the complex of proteins including the core DNA polymerase 111 and several auxiliary proteins which acts as the natural replicative enzyme in E . coli. DNA polymerase I11 is the name used for the catalytic core of the holoenzyme. DNA polymerase I11 can fill in short gaps in nu- clease-treated DNA but cannot replicate natural template probes such as G4, M13, or 41x174 without the addition of holoenzyme auxiliary subunits. DNA polymerase III* is a complex form of DNA polymerase I11 which contains some holoenzyme subunits, but re- quires the addition of /3 to reconstitute holoenzyme activity.

Two forms of DNA polymerase I11 have been isolated that are subassemblies of holoenzyme subunits. A core DNA po- lymerase I11 has been purified that is composed of the a, t, and 6‘ subunits of the DNA polymerase I11 holoenzyme (McHenry and Crow, 1979). Alone, the a subunit of this core complex has at least a limited capacity to synthesize DNA (Spanos et al. 1981). The DNA polymerase III* (Wickner et al., 1973) form of the holoenzyme requires only p to reconsti- tute holoenzyme activity in single strand phage assays. Struc- turally, it is less characterized than polymerase 111, but is known to contain at least the y and S holoenzyme subunits, in addition to the core DNA polymerase I11 (McHenry and Kornberg, 1977).

The functions of the auxiliary holoenzyme subunits are not yet clear. The p subunit is required for the DNA polymerase I11 holoenzyme to form an ATP (or dATP)-dependent initia- tion complex with primed G4 DNA (Johanson and McHenry, 1980). Once this complex is formed, DNA synthesis becomes resistant to anti-p immunoglobulin G; nevertheless, ,8 is still part of the complex. Formation of an initiation complex ap- parently sterically blocks anti-immunoglobulin G from bind- ing the p subunit (Johanson and McHenry, 1981).2 Other work has suggested that elongation factor I11 or dna Z protein, when separately isolated, can transfer Factor I (p) to a primed DNA single strand (Wickner, 1976). An evaluation of the processivity of DNA polymerase I11 and the DNA polymerase 111 holoenzyme demonstrate that the auxiliary subunits in- crease the number of nucleotides inserted per association event from 10-15 to greater than 5000 (Fay et al. 1981).

Further characterization of DNA polymerase III* and ho- loenzyme have been frustrated by their lability and the pos- sible lack of assays for components which may serve important functions in the replication of the complex E. coli chromo- some, but not in the relatively simple in vitro phage replica- tion assays. In this paper, I report the purification and char- acterization of a well defined polymerase form which is inter- mediate in function and structure between DNA polymerase I11 and DNA polymerase III*. Its purification has led to the assignment of a new holoenzyme subunit, T , and a partial understanding of this subunit’s contribution to the holoen- zyme reaction.

MATERIALS AND METHODS

Buffers-These were: T (50 mM Tris.HCI (pH 7.5), 20% glycerol, 1 n “ , EDTA, 5 mM dithiothreitol); PC (50 m~ Tris.HC1 (pH 7.5). 30% glycerol, 1 mM EDTA, 5 mM dithiothreitol); HA (25% glycerol, 50 n” Imidazole.HC1 (pH 6.60, 1 M KCI, 5 m~ dithiothreitol); A (potassium phosphate (pH 6.5) (concentration varies and will be designated), 25% glycerol, 1 m~ EDTA, 5 mM dithiothreitol); enzyme dilution buffer (50 m~ Tris.HCI (pH 7.5), 20% glycerol, 10 mM dithiothreitol, 0.1 m~ EDTA, 0.2 mg/ml of bovine serum albumin).

K. Johanson and C. McHenry, unpublished results.

2657

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2658 DNA Polymerase 111' Chromatography Supports-Phosphocellulose (P11) was obtained

from Whatman; hydroxylapatite was manufactured by Clarkson Chemical, Williamsport, PA; Sephacryl S-300 (Superfine) was ob- tained from Pharmacia; AcA34 was purchased from LKB.

Activated Salmon Sperm DNA Assay-This assay was performed essentially as described (McHenry and Crow, 1979). The reaction was initiated by the addition of DNA polymerase 111 to a solution (30 p l ) containing 33 mM 4-morpholinopropanesulfonic acid (pH 7.0); 17 mM dithiothreitol; 133 dCTP, GTP, and dATP 50 PM [3H]dTTP (-70 cpm/pmol); 13.3 nm MgClz, 10% ethanol, and 167 pg/ml of activated salmon sperm DNA. In all assays, if necessary, the enzyme was diluted with enzyme dilution buffer. The reaction was stopped after 5 min. One unit in this and all other assays is defied as the amount of enzyme catalyzing the incorporation of 1 pmol of deoxynucleotide (total)/min. Acid-insoluble radioactivity was determined as described (McHenry and Crow, 1979). The DNA polymerase 111' purification (Table I) was monitored by this procedure and peak assignments were only c o n k e d by the fd assay because the activated salmon sperm DNA assay gave more precise and linear results, especially when dilute enzyme was assayed. Also, the levels of activity tend to fluctuate with interconversion of enzyme forms when the fd assay is used.

Primed fd DNA Assay-This assay was performed as described for the activated salmon sperm DNA assay except that ethanol was deleted, spermidine. HC1 (5 mM) was added, the assay was incubated for 10 min, and primed fd DNA (50 p in total nucleotide) was used.

G4 Assay-This assay which uses single-stranded DNA binding protein encoated G4 single-stranded DNA primed by dna G primase as a template was performed as described (McHenry and Kornberg, 1977; Johanson and McHenry, 1980).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis- Slab gel electrophoresis was conducted in a 7.5 -+ 17.5% acrylamide gel (5% stacking gel) in 0.1% SDS3 as described (Laemmli, 1970; McHenry and Crow, 1979). Protein was detected by Coomassie blue staining as described (McHenry and Crow, 1979). Densitometry was performed at 600 nm on a Helena Quick Scan R and D scanning densitometer.

DNA-Salmon sperm DNA was activated as described (Livingston et al. 1975; McHenry and Crow, 1979). Primed fd DNA was a generous gift of Phil Fay and Dr. Robert Bambara of the University of Roch- ester. It was prepared as described (Fay et al., 1981).

Proteins and Enzymes-Unless specified otherwise, polymerase 111 fractions used in the reported experiments were: DNA polymerase 111 (Fraction VI; McHenry and Crow, 1979), DNA polymerase 111' (Fraction VI concentrated), DNA polymerase III* (Sephacryl S-300 fraction; Fay et al., 1981; specific activity 1 X lo6 units/mg), and the DNA polymerase 111 holoenzyme (Fraction V, McHenry and Korn- berg, 1977). p (Fraction IV, Johanson and McHenry, 1980) and single- stranded DNA binding protein (Meyer et al. (1980)) were prepared as described.

RESULTS

Purification of DNA Polymerase 111' DNA polymerase 111' was purified 15,000-fold from E. coli

HMS-83 (Table I). All operations, unless noted, were per- formed at 0-4 "C. Enzyme fractions obtained from chromat- ographic procedures which contained at least 50% of the peak activity were combined.

Cell Growth-E. coli HMS 83 (Campbell et al., 1972) was grown to late log phase at 37 "C (Asn, = 6) in 188 liters of media containing 1.88 kg of yeast extract, 1.88 kg of glucose, 1.9 g thiamine, 9.4 g of thymine, 2.5 kg of KzHP04 3H20,350 g of KH2P04 with high aeration (280 liter/min, 15 p . 4 . The pH was maintained between 7.0 and 7.4. Cells were harvested within 1 h in a continuous flow centrifuge after passing through a %-foot copper coil submerged in ice to cool the cells to 21 "C. Cells were weighed, resuspended in an equal weight of 50 mM Tris-HC1 (pH 7.5), 10% sucrose, and immediately poured into liquid N2.

Preparation of Extracts-Frozen cells (3 kg of cell weight, 6 kg total) were thawed in a solution containing 50 lIlM Tris. HC1 (pH 7.5), 10% sucrose, 0.1 M NaC1, and 15 II~M spermidine

The abbreviation used is: SDS, sodium dodecyl sulfate.

TABLE I Purfication of DNA polymerase III' Fraction Total units Specific activity

xIo-3 x I r 3 units/mg

I. Lysate supernatant 17,000 protein

0.12 11. Ammonium sulfate 13,000 3.5

111. Phosphocellulose I 6,400 55 IV. Hydroxylapatite 4,200 100

concentrate 1,700

Total 620 Pol 111' peak 220 110 Concentrate 200

Total 140 Pol 111' peak 100 1700

V. Phosphocellulose I1

VI. Gel filtration

(0.2 g of cells/ml, final concentration). The pH was adjusted to 7.6 to 7.7 with 2 M Tris base, and egg white lysozyme (0.2 mg/ml) was added. This mixture was transferred to 250-ml centrifuge bottles, incubated at 4 "C for 1 h, and then heated in a 37 "C bath for 4 min with gentle inversion of the bottles each minute. The cells were centrifuged (23,000 X g, 0 "C, 1 h), resulting in a 12.5-liter supernatant (Fr I).

Ammonium Sulfate Fractionation-To Fr I was added 0.226 g of (NH4)2S04/ml of Fr I (0.206 g/ml, final concentra- tion). After 30 min at 0 "C, the resulting suspension was centrifuged (23,000 X g, 0 "C, 40 min). The precipitate was washed successively with Buffer T + 0.1 M NaCl to which 0.20 and 0.17 g of (NH4)2S04 had been added to each milliliter (1/ 8 Fr I volume, then 1/50 Fr I volume, respectively). Each wash was followed by centrifugation (31,000 X g, 0 "C, 30 min), resulting in a final pellet which was dissolved in 120 mi of Buffer PC + 25 nm NaC1. This solution was clarified by centrifugation (31,000 X g, 0 "C, 40 min) and dialyzed versus 10 liters of Buffer PC + 25 nm NaCl overnight, resulting in Fr I1 (112 ml).

Phosphocellulose I-Dialyzed Fr I1 was diluted to a con- ductivity between that of Buffer PC + 25 -+ 35 mM NaCl with 10 mM Tris, pH 7.5, 30% glycerol, 5 mM dithiothreitol. (Cau- tion: do not dilute Fr I1 to a lower conductivity.) The resulting solution (183 m l ) was applied to a 667-ml phosphocellulose column equilibrated with Buffer PC + 25 mM NaC1. The activity was eluted with a six-column volume gradient (Buffer PC + 40 m NaCl + Buffer PC + 300 II~M NaC1) at a flow rate of one-column volume/h. The enzyme was immediately assayed and fractions pooled resulting in Fr I11 (1100 ml).

Hydroxylapatite-To Fr I11 was added 0.1 volumes of 0.5 M imidazole.HC1 (pH 6.8), 0.01 volumes of 0.5 M potassium phosphate (pH 6.5), and 84 g of KC1 (final concentration, 1 M KC1). This solution was applied to a 60-ml hydroxylapatite column (10-column volume/h) equilibrated with Buffer HA + 5 nm potassium phosphate. The activity was eluted with a 10-column volume gradient (Buffer HA + (5 mM + 100 mM potassium phosphate), one-column volume/h). Fractions were pooled to yield Fr IV (137 ml). An equal volume of saturated (NH4)&04 was added to Fr IV, stored overnight at 0 "C, centrifuged (18,000 X g, 0 "C, 1.7 h) in a swinging bucket rotor, and the resulting pellet was redissolved in 1.5 ml of Buffer A (40 nm potassium phosphate) to yield concentrated Fr IV.

Phosphocellulose 11-Concentrated Fr IV was dialyzed ver- sus 500 ml of Buffer A (40 nm potassium phosphate) for 5 h. The resulting turbid solution was adjusted to the conductivity of 40 mM potassium phosphate by the addition of 25% glycerol, 5 nm dithiothreitol (3.7 ml, final volume). This solution was applied to a 14-ml phosphocellulose column equilibrated in Buffer A (40 nm potassium phosphate). The column was

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DNA Polymerase 111’ 2659

washed with one-column volume of equilibration buffer and eluted with a 15-column volume gradient (Buffer A (40 mM + 250 mM potassium phosphate), one-column volume/h). Two polymerase peaks eluted (Fig. 1). The first peak was DNA polymerase I11 and the second DNA polymerase 111’ (see following sections for characterization). The ratio of these two polymerase forms varied between 2:l (here) and 1:2 in various preparations. The DNA polymerase 111’ peak was pooled to yield Fr V (23 ml). The DNA polymerase I11 peak can be purified separately by a method similar to that de- scribed below for DNA polymerase III’. Fr V was concentrated in a collodion bag (25,000-dalton cutoff) uersus Buffer A (100 mM potassium phosphate) to yield concentrated Fr V (0.28 ml) .

Gel Filtration-Fr V (200 p1) was applied to an 8 5 4 Sephacryl S-300 column equilibrated in Buffer A (100 mM potassium phosphate). Fractions (1.0 m l ) were collected at a rate of 3.5 ml/h. Active DNA polymerase 111’ fractions were pooled to yield Fr VI (5 ml). For some studies, Fr VI was concentrated in a collodion bag apparatus as described for step V, except that carrier bovine serum albumin was added (1 mg/ml, final concentration).

Fr VI is stable for at least 1 year when rapidly frozen in liquid NP and stored at -80 “C. Intermediate fractions in the aforementioned preparation are stable and can be stored at the ammonium sulfate pellet stages. It is important to run the initial phosphocellulose column and star t the gradient on the hydroxylapatite column in the same day since the enzyme is labile at this stage.

Physical Characterization

SDS-Polyacrylamide Gel Electrophoresis-DNA polym- erase 111’ (Fr VI, 10 pg) was denatured and electrophoresed on an SDS-polyacrylamide gel. Four major protein bands of 140,000, 83,000, 25,000, and 10,000 daltons were present (Fig. 2). The largest and two smallest components are subunits of the core DNA polymerase 111. The fourth component (83,000 daltons) is not part of the core polymerase and is termed T. A densitometric scan indicates the ratio of a : ~ to be 1:l.l. The smaller subunits, E and 8, vary between 1 and 2 in different preparations. Due to the variance in dye binding to proteins of very different molecular weight, I will withhold assignment of E and B stoichiometries. The densitometric scan also indi-

Fraction

FIG. 1. Separation of DNA polymerase In from DNA polym- erase 111’ by phosphocellulose chromatography. The column was that described for the phosphocellulose I1 step. The gradient was begun at fraction 4 and ended at fraction 56. DNA polymerase 111 assays (0) on activated salmon sperm DNA and DNA polymerase 111’ (0) assays on primed fd DNA were as described under “Materials and Methods.”

cates the DNA polymerase 111’ preparation to be 90% pure. Co-chromatography of cu, T, 6, and 8 with DNA Polymerase

ZII’-a, T, E , and 8 chromatograph with DNA polymerase 111’ on both phosphocellulose and gel filtration. It is demonstrated (Fig. 3) that a, E, and 8 increase and decrease in parallel with polymerase I11 activity upon phosphocellulose chromatogra- phy; yet, T increases and decreases with only the DNA polym- erase 111’ form. I conclude that DNA polymerase 111’ is a complex of DNA polymerase I11 and T , and that this associa- tion changes the physical properties of the enzyme such that it is separable from DNA polymerase 111.

Presence of T Subunit in the DNA Polymerase 111 Holoen- zyme-The T subunit of DNA polymerase 111’ is present in highly purified DNA polymerase I11 holoenzyme (Fig. 4). T

co-migrates with an 83,000-dalton component present in all holoenzyme preparations. The holoenzyme fraction used (Fig. 4) was the most highly purified preparation available; it was purified by a procedure (McHenry and Kornberg, 1977) mark-

FIG. 2. Densitometric scan of SDS-polyacrylamide gel. DNA polymerase 111’ (Fr VI; 10 pg) was denatured and subjected to elec- trophoresis in a 7.5 + 17.5% acrylamide gel as described under “Materials and Methods.” The subunits of DNA polymerase 111’ are labeled.

12 16 18 20 22 24 26 29 32 34 36 38 40 45

FIG. 3. Correlation of polymerase components to activity on a phosphocellulose column. Aliquots (200 pl) of fractions from a phosphocellulose column were denatured and subjected to electro- phoresis in a 7.5+ 17.5% acrylamide gel as described under “Materials and Methods.” Fraction 20 and 36 corresponded with the DNA polymerase 111 and polymerase 111’ peaks, respectively. Aliquots applied contained the following units in the activated salmon sperm DNA assay: lane 12, 0.1 units, lane 16, 0.4 units, lane 18, 1.9 units, lane 20, 8.5 units, lane 22, 3.0 units, lane 24, 1.2 units, lane 26, 0.6 units, lane 29, 0.2 units, lane 32, 0.3 units, lane 34, 1.5 units, lane 36, 6.6 units, lane 38, 2.6 units, lane 40, 1.0 units, and lane 45, 0.1 units. The phosphocellulose column was run as described for the phospho- cellulose I1 column in this paper except that some contaminants were first removed by filtration through AcA34 to simplify the gel pattern. Fr IV (concentrated; 1.2 ml) was applied to an AcA34 column (85 ml) equilibrated with Buffer A (20 m~ potassium phosphate) + 0.1 M KCI. AU fractions containing DNA polymerase I11 or polymerase 111‘ activity were combined (14 ml, 70% yield), and diluted with 3 volumes of Buffer A (20 m~ potassium phosphate) before application to the phosphocellulose column.

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2660 - DNA Polymerase 111’

FIG. 4. SDS-acrylamide gel of DNA polymerase III holoen- zyme. The subunits common to both the holoenzyme and DNA pol.ymerase 111‘ are labeled. The holoenzyme used (15 pg) had been sedimented through a glycerol gradient after Fraction V (McHenry and Kornberg, 1977).

edly different from that used for DNA polymerase 111’. Based upon this information and the functional information which follows, T is assigned as a subunit of the DNA polymerase I11 holoenzyme. Thus, DNA polymerase 111’ is a subassembly of four holoenzyme subunits.

Determination of the Molecular Weight of DNA Polymer- ase ZZZ’-The molecular weight of DNA polymerase 111’ was calculated from the Stokes radius and sedimentation coefi- cient by the method of Siege1 and Monty (1966). Gel filtration indicates that DNA polymerase 111’ is much larger than DNA pol-ymerase I11 (Fig. 5A) . A plot of its elution position relative to standards (Fig. 5 0 yields a Stokes radius of 85 A. Although the starting material for this experiment contained no free DNA polymerase 111, some dissociated during filtration (Fig. 5A) and eluted in the same position as DNA polymerase I11 filtered in a separate experiment (Fig. 5B).

Sedimentation of DNA polymerase 111’ in a glycerol gra- dient also indicated the instability of this enzyme form. Two peaks are observed in a glycerol gradient (Fig. 6A), one of which corresponds to the single peak resulting from DNA polymerase I11 sedimentation (Fig. 6B). The more rapidly sedimenting peak which is assigned to DNA polymerase 111’ on the basis of this information and a specific assay described in later sections has a sedimentation coeficient of -11.3 S (Fig. 6 0 .

From these data, a M, = 160,000 is calculated for DNA polymerase I11 and 410,000 for DNA polymerase III’. Al- though approximate due to the broad peaks obtained, and the assumption that the partial specific volume is the same in both enzyme forms, these data indicate that the mass of DNA polymerase 111’ is greater than twice that of DNA polymerase 111. Taken together with the 1:l stoichiometry between a and T, the data is most consistent with DNA polymerase 111’ containing two core DNA polymerase I11 assemblies and two r subunits.

Characterization of Polymerase Activity Activity on Primed fd DNA in the Presence of Spermi-

dine-DNA polymerase 111’, like DNA polymerase I11 holoen- zyme and DNA polymerase III* (Wickner and Kornberg, 1974; Wickner et al., 1973) can replicate primed fd or M13 DNA in the presence of spermidine (Fig. 7). DNA polymerase 111’ and DNA polymerase I11 exhibit approximately the same activity in this assay; the DNA polymerase I11 holoenzyme is significantly more active. The low activity of DNA polymerase I11 observed on these templates is strongly inhibited by 3 mM or higher concentrations of spermidine (Fig. 7). This provides a functional assay for distinguishing DNA polymerase 111’ from DNA polymerase I11 during chromatographic manipu- lations. Although two peaks of polymerase activity assayed on activated salmon sperm DNA appear during the phosphocel- lulose I1 or gel filtration steps, only the DNA polymerase 111’ peak is active in the fd assay. This confirms the assignment of the DNA polymerase 111’ peak which was originally based upon physical properties and gel electrophoresis.

Effect of Single-stranded DNA Binding Protein upon Var- ious Forms of DNA Polymerase ZZZ-DNA polymerase 111’ can be distinguished from DNA polymerase 111’ by substitut- ing single-stranded DNA binding protein for spermidine in the primed fd assay. This protein stimulates DNA polymerase III*, but not DNA polymerase 111’ (Fig. 8 ) . The DNA polym- erase I11 holoenzyme is slightly stimulated above its preexist- ing high activity, and the low activity of DNA polymerase I11 is inhibited by single-stranded DNA binding protein (Fig. 8 ) .

Combining this information with the knowledge that only holoenzyme (or DNA polymerase III* in the presence of the p subunit) is active in the natural G4 replication system, all known forms of DNA polymerase I11 can be distinguished by functional assays (Table 11).

DISCUSSION

I have purified DNA polymerase 111’ 15,000-fold from E. coli HMS-83, a pol A, pol B mutant. The enzyme is at least 90% pure based upon a densitometric scan of SDS-gels of Fraction VI. The purification of DNA polymerase 111’ is similar to that of DNA polymerase I11 (McHenry and Crow, 1979). The critical difference is the concentration step follow- ing hydroxylapatite chromatography. If Fr IV is concentrated to 2.5 X IO6 units/ml, a mixture of DNA polymerase I11 and 111’ is observed upon subsequent phosphocellulose chromatog- raphy (Fig. 1) or gel filtration (data not shown). The propor- tion of DNA polymerase 111’ has been found to vary between 70 and 30% of total DNA polymerase 111. Collodion bag

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DNA Polymerase III' 2661

I

FIG. 5. Gel filtration of DNA polymerase HI' and determi- pl was applied to the same column as described in A. DNA polymerase nation of Stokes radius. A, the column was that described for the I11 (0) and polymerase 111' (not present) was assayed as in A. C, a gel filtration step of the DNA polymerase 111' purification. Fractions plot to determine the Stokes radius of DNA polymerase 111 and DNA were 1.0 ml. DNA polymerase I11 (0) and DNA polymerase 111' (0) polymerase III'. Standards were (X) thyroglobulin, (A) jack bean assays were as described in Fig. 1. B , DNA polymerase I11 was urease, (0) catalase, (0) yeast alcohol dehydrogenase, (M) bovine obtained from the first peaks of the phosphocellulose I1 column. It serum albumin. The void volume (30 ml) was determined using phage was concentrated to 400 p1 as described for DNA polymerase III'; 300 P22.

centrated as described in Fig. 4) was treated as described for DNA polymerase 111' in A. C, a plot to determine the sedimentation coefficient of DNA polymerase I11 and DNA polymerase 111'. Stand- ards which were sedimented in a separate tube in the same rotor were: (A) urease, (X) P-galactosidase, (0) catalase, (0) yeast alcohol dehydrogenase, (M) bovine serum albumin. Bovine serum albumin sedimented in the same position in the experiments described in A, B, and the standard tube. Arrows indicate the direction of sedimen- tation.

i o 20 4 i

Frasflm

FIG. 6. Determination of the sedimentation coefficient of DNA polymerase III. A, sedimentation of DNA polymerase 111'. DNA polymerase 111' (Fr V concentrated, 50 pl) was mixed with bovine serum albumin (75 pl, 1 mg/ml) and was sedimented through a 25 + 40% glycerol gradient in 100 m~ potassium phosphate (pH 6.8), 5 m~ dithiothreitol for 14 h at 56,000 rpm in a Beckman SW 60 rotor a t 2 "C. Fractions (50 total) were dripped from the bottom of the tube. DNA polymerase I11 (0) and DNA polymerase 111' (0) were assayed as described in Fig. 1. B, sedimentation of DNA polymerase ILL. DNA polymerase I11 (50 pl, phosphocellulose 11, first peak, con-

I , "I 0 1 2 3 4 S 6

Spermidine (mM)

FIG. 7. Effect of spermidine on various forms of DNA polym- erase ID. The spermidine concentration was varied in the primed fd assay described under "Materials and Methods" using (0) DNA polymerase I11 holoenzyme, (0) DNA polymerase HI*, (0) DNA polymerase 111', and (A) DNA polymerase 111 as an enzyme source. All tubes contained 125 units of activity measured in the activated salmon sperm DNA assay.

concentration has also been successful at this point. T appears to be loosely associated with pol I11 and chromatography under dilute enzyme concentrations leads to its dissociation and separation. DNA polymerase HI', obtained by the purifi- cation reported herein, contains four subunits: a, T, e, and 0 of

Singie4tranded DNA Binding Protein (ul)

FIG. 8. Effect of single-stranded DNA binding protein on the various forms of DNA polymerase III. Spermidine was omitted and the single-stranded DNA binding protein concentration was varied in the assay described in Fig. 7. The single-stranded DNA binding protein solution used in this experiment was 0.9 mg/ml.

140,000, 83,000, 25,000, and 10,000 daltons, respectively. The ratio of a to T is 1:1, based upon densitometric scans of stained gels. The assignment of T as a subunit of DNA polymerase 111' and holoenzyme is based upon the following physical evidence in addition to the functional data discussed below. (i) T has been isolated bound specifically to DNA polymerase 111. When T is associated, the polymerase binds to phospho- cellulose more tightly and becomes larger. Upon rechroma-

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2662 DNA Polymerase 111’

TABLE I 1 Functional distinctions between DNA polymerase 111 forms

Assays were performed as described under “Materials and Meth- ods” except that the volume and total quantity of components was twice that reported for the activated salmon sperm and G4 DNA assay. The primed fd assay contained 5 m~ spermidine or 1.8 pg of single-stranded DNA binding protein (ssb). /3 (20 units, Fr VI; Johan- son and McHenry, 1980) was added where designated to the G4 assay. Results are exuressed in total Dmol nucleotide incomoration.

Activated Primed fd G4 Form salmon

sperm Spermi- =,, DNA dine

- +P

Polymerase I11 570 5 11 2 2 Polymerase 111’ 630 67 62 6 10 Polymerase I I I * 660 48 143 28 470 Holoenzvme 620 190 180 294 380

____

tography, some DNA polymerase 111’ dissociates to yield free DNA polymerase I11 but not vice versa. (ii) DNA polymerase I11 holoenzyme, which is purified by a drastically different procedure, contains T in a 1:l ratio with the a subunit (Fig. 4). The 83,000-dalton component was observed in previous hol- oenzyme preparations (McHenry and Kornberg, 1977), but was not assigned as a subunit at that time due to the lack of additional evidence. (iii) Immunoprecipitation of holoenzyme with anti+ IgG precipitates a and T in a 1:l ratio (Johanson and McHenry, 1980; McHenry, 1980). In the absence of asso- ciated p, neither a nor T are precipitated by this antibody.

In addition to the aforementioned physical evidence, func- tional studies also suggest T to be a subunit of the DNA polymerase I11 holoenzyme. When T is associated with DNA polymerase 111, its functional properties become more holoen- zyme-like. All polymerase forms can use duplex DNA contain- ing short gaps as a template. However, early holoenzyme studies demonstrated that holoenzyme could utilize a long single-stranded DNA template in the presence of spermidine and that DNA polymerase I11 could not (Wickner and Korn- berg, 1974). When I used an fd DNA template containing -3 short (50 nucleotide) primers/circle, I found polymerase I11 to be inert in the presence of spermidine, but holoenzyme, DNA polymerase III*, and DNA polymerase 111’ were active. On this template, if spermidine was replaced by single-stranded DNA binding protein, only DNA polymerase III* and holoen- zyme were stimulated. Only DNA polymerase I11 holoenzyme was active by itself in the G4 assay. Additionally, it has been demonstrated (Fay et al., 1981) that the processivity of DNA polymerase I11 and DNA polymerase I11 holoenzyme differ, under optimal conditions, by at least 300-fold. It has recently been found4 that the processivity of DNA polymerase 111’ is intermediate between that of DNA polymerase I11 and hol- oenzyme and that spermidine increases the processivity of DNA polymerase 111’, like holoenzyme. Thus, there is a gra- dient of functional properties that parallels the structural complexity of the polymerase forms.

I have also examined the requirement of T to reconstitute holoenzyme activity on the G4 template from its known components: pol 111, p, and the y-6 complex. I see no require- ment (data not shown); holoenzyme is reconstituted with equal efficiency from DNA polymerase I11 and DNA polym- erase 111’. This result might be explained if T served a function in vivo for the replication of the complex E. coli chromosome that is not important in vitro for the replication of a relatively simple single-stranded template. The molecular weight stud- ies described below may suggest one such function. It must be remembered that the DNA polymerase I11 holoenzyme was

P. Fay, K. Johanson, C. McHenry, and R. Bambara (1982) J. Biol. Chem, in press.

isolated by two criteria (McHenry and Kornberg, 1977): (i) it was assayed by its functional requirement in the G4 and other assays, and (ii) it was required to be an intact physical complex. Purification steps which led to its disruption were not used (i.e. phosphocellulose). Thus, subunits may have been isolated, by virtue of their requirement for an intact complex, which are not required for reconstitution of G4 replicative activity upon resolution of that complex.

The shift in the native moleculm weight of DNA polymer- ase I11 from 160,000 to greater than 400,000 when converted to DNA polymerase 111’ taken together with the subunit ratio of a : ~ being 1:l suggests that DNA polymerase 111’ contains two core DNA polymerase I11 assemblies and two subunits of T. Arthur Kornberg recently made the interesting hypothesis that a single holoenzyme molecule might replicate both strands of a duplex DNA molecule at a natural replication fork simultaneously (Arai and Kornberg, 1981).5 This could either be due to the a subunit having two active sites or holoenzyme containing two a subunits. The data in this paper support the latter posisbility. T , when bound to DNA polym- erase 111, may allow the polymerase to dimerize, providing a core upon which other polymerase auxiliary subunits assem- ble. The functional changes in polymerase activity reported in this paper could be due to a direct participation of T in the catalytic reaction or caused by the ability of T to bind polym- erase I11 and stabilize a holoenzyme-like conformation in its activity site.

The assignment of T brings the total number of DNA polymerase I11 holoenzyme subunits to seven. An SDS-gel of the most highly purified holoenzyme available (Fig. 4) con- tains at least 13 bands, all of which co-sediment in a glycerol gradient? Further work will be directed toward removing the remaining six unassigned components or establishing their function in the replicative reaction.

Acknowledgments-I wish to acknowledge the excellent technical assistance of Reginald Jones, Weldon Crow, Gary Coleman, and David Cooper. I wish to thank Dr. Ralph Meyer of the University of Cincinnati for his helpful discussions throughout the course of this work. I am grateful to Phillip Fay and Dr. Robert Bambara of the University of Rochester for their generous gift of primed fd DNA.

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C S McHenrya subunit of the DNA polymerase III holoenzyme.

Purification and characterization of DNA polymerase III'. Identification of tau as

1982, 257:2657-2663.J. Biol. Chem. 

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