A rapid procedure for isolation of large quantities of Escherichia coli DNA polymerase I utilizing a lambdapolA transducing phage.

DNA polymerase I produced by infection of Escherichia coli K12 with the specialized transducing phage lambdapolA has been purified by a simplified procedure and shown to be identical with the enzyme produced by uninfected E. coli in all aspects which have been examined. The abundance of the enzyme in infected cells and the ease with which it may be purified will simplify the study of the enzyme's physical and chemical characteristics. In addition, the enzyme is now much more readily available for use as an analytical tool in nucleic acid sequence and structure studies.


DNA polymeraee
I produced by infection of Escherichia coli K12 with the specialized transducing phage XpolA has been purified by a simplified procedure and shown to be identical with the enzyme produced by uninfected E. coli in all aspects which have been examined.
The abundance of the enzyme in infected cells and the ease with which it may be purified will simplify the study of the enzyme's physical and chemical characteristics.
In addition, the enzyme is now much more readily available for use as 'an analytical tool in nucleic acid sequence and structure studies.
The isolation of a specialized transducing phage h derivative carrying the Escherichia colipolA gene (1) has made possible the isolation of large quantities of the enzyme DNA polymerase I. This enzyme is generally useful for many experiments in nucleic acid chemistry, and we describe here a simplified procedure by which the enzyme may be purified from phageinfected cells or cells of a heat-induced Xc1857 lysogen. Biochemical and enzymological characterization indicates that this material is identical with that previously isolated from uninfected E. coli (2)' in which it is normally present in relatively low abundance.

EXPERIMENTAL PROCEDURES
Materials-E. coli and bacteriophage strains are described in Table I. Transducing phage NM825 which lacks the h attachment site has been described previously (1). The integration proficient derivative phage NM857 was derived from phage NM825 by appropriate crosses. Lysogens of phage NM857 were isolated by infecting E. coli JG108 with this phage and spreading infected cells on nutrient plates at 32°C. The colonies which appeared were further tested to verify lysogeny on the basis of resistance to hc1' and sensitivity to himm@' phage stocks in cross-streak tests. Bacteria were grown in L broth (3) or in superbroth (4) as indicated.

Intracellular
Amplification of DNA Polymerase I Levels-The DNA polymerase I levels attained within the cells of E. coli hpolA lysogens induced by heat shock or within E. coli cells infected with transducing phage vary depending upon the metabolic condition of the cells. Both methods were tested for optimal conditions of enzyme production.
Prophage Induction-The E. coli strain CM4935, lysogenized with ApolA phage NM857, was grown from a single colony in overnight culture in 500 ml of L broth at 32°C. This culture was inoculated into 5 liters of superbroth in a 6-liter New Brunswick fermenter jar at 32°C and grown with vigorous aeration until the culture reached a turbidity corresponding to 6 to 8 x IO8 cells/ml. At this time the temperature of the culture was adjusted to 42"C, held for 25 min, and then cooled to 37°C and maintained at this level for the remainder of the phage induction. Samples of the culture were removed at 30min intervals and assayed for DNA polymerase I (1). Maximal amplification of the enzyme occurs in 4 to 5 h after phage induction and amounts to 20-to 40-fold that found in wild type nonlysogenized E. coli K12. The extent of the amplification is dependent on the richness of the medium, the manner of aeration, and the density of the cells at the time of induction. Cells were harvested by centrifugation in the Sorvall GSA rotor (10,000 x g for 15 min), and the cell pellets were frozen before lysis and enzyme extraction. Yields of cells are 5 to 8 g (wet weight)/liter by this protocol. Induction of the cells at higher cell densities will give larger yields of cells but generally lower specific activities of DNA polymerase I per cell.
Infection with Nonlysogenic Transducing Phage-Higher yields of DNA polymerase I may be obtained from a culture of E. coli which are grown in broth and infected with the integration-deficient XpolA transducing phage previously described (1). Polymerase production controlled by this phage is influenced by gene dosage, and maximal yields are obtained by infecting the bacteria at a multiplicity of 10 to 15 phage/ cell. Yields of polymerase obtained by this method are about 2-fold higher than those obtained by induction of the lysogen.
Phage NM825 was propagated using the supF strain Ymel. Infected cells were diluted in soft agar and spread over L agar plates. The phage particles were harvested after 8 to 12 h by scraping the plates and removing agar and cellular debris by centrifugation.
This method yielded phage titers of 3 to 8 X 10" infectious phage/ml. E. coli strain JG108 was grown from a single colony in overnight culture in 500 ml of L broth. This culture was diluted into a further 2 liters of L broth containing 0.2% maltose in a 6-liter New Brunswick fermenter jar and grown through two generations at 37°C to a cell density of 1.2 to 1.6 x 10' cells/ml. The cells were then collected by centrifugation at 10,000 X g for 10 min, resuspended in 100 ml of 0.01 M MgCl*, 0.01 M Tris, pH 7.6, and mixed with phage at a multiplicity of infection of 10 phage/cell. After 15 min of adsorption at 37'C the culture was diluted into 6 liters of super broth at 37°C and vigorously aerated for an additional 4 to 6 h. Cultures were sampled, and polymerase levels were analyzed as in the induction experiment. Yields of polymerase were consistently greater than those observed following derepression of the lysogen and approximated those observed previously in smaller cultures (1). The final cell mass was harvested by centrifugation as the heat-induced culture had been. Total quantities of cells were approximately 5 to 8 g wet weight/liter of final culture with variation from batch to batch. The pellets were frozen before lysis and enzyme extraction.
Cell Lysis and Enzyme Purification-A modified DNA polymerase I preparation has been devised for use with cells containing the phage-amplified enzyme. This preparation utilizes the polyethyleneimine precipitation procedure fist devised for RNA polymerase preparation by Jendrisak and Burgess (13) and modified by Panasenko et al. (14) for preparation of DNA ligase. All of the succeeding steps were carried out in the cold room or at ice bath temperatures.
Frozen cell pellets were thawed and suspended in 50 mM Tris, pH 7.6, 1 mM dithiothreitol (175 g of cells to 470 ml of buffer). Cell lysis was completed, and the viscosity of this preparation was reduced by passage through a French pressure cell. Bacterial debris was removed by centrifugation at 10,600 x g for 10 min, and the supernatant was transferred to a beaker. Aliquots of this supernatant were removed and titrated with increasing amounts of 10% solution of Poiymin P in water (pH 7.6). Precipitates were removed by centrifugation at 15,900 x g for 10 min in the cold, and the supernatants were assayed for DNA polymerase I activity in the standard assay. After optimal conditions for precipitation were established (0.9% final Polymin P) the entire cell extract was precipitated by gradual addition of 61.9 ml of the 10% Polymin P solution to the 625-ml volume of extract. The precipitate formed was collected by centrifugation at 15,000 x g for 10 min.
The Polymin P pellet was resuspended in 210 ml of 20 mM potassium phosphate, pH 6.5, 2 mu EDTA, 1 mM dithiothreitol, 0.2 M ammonium sulfate by gentle low speed stirring in L Waring Blendor in the cold. The resuspension was then clarified by centrifugation at 15,000 x g for 10 min, and the supernatant was removed and saved. The pellet was again extracted with 200 ml of the same buffer, and the supernatants of the two extractions were pooled.
Solid ammonium sulfate was gradually stirred into this supematant to bring the final ammonium sulfate concentration to 40% saturation at zero degrees (15). When all solid was dissolved the suspension was allowed to stand for 1 h in the cold room, and the precipitate was removed by centrifugation at 15,000 X g for 15 min. Crystalline ammonium sulfate was gradually added to this supernatant with stirring until a final ammonium sulfate concentration of 85% saturation was reached. This suspension was allowed to stand overnight in the cold room before the precipitate was collected by centrifugation at 15,000 X g for 15 min.
The precipitated protein from this ammonium sulfate cut was then dissolved in 200 mM potassium phosphate, pH 6.5, 10 mM mercaptoethanol, giving a final volume of 86 ml. This DNA Polymerase I from ApolA Transducing Phage solution was passed directly through a column (5 cm (diameter) x 15 cm) of DEAE-cellulose equilibrated in 200 mM potassium phosphate, pH 6.5, 10 mM mercaptoethanol and eluted with the same buffer. Fractions of 10 ml were collected, and the absorbance of the fractions at 280 nm and 260 nm was checked. Fractions with a 280/260 ratio of greater than one were pooled (total volume = 315 ml; net 280/260 ratio = 1.21) and the protein reprecipitated by adding 173 g of solid ammonium sulfate gradually while stirring in the cold. The final precipitate was collected by centrifugation at 15,000 x g for 25 min. This precipitate was then dissolved in 73 ml of 100 mM potassium phosphate, pH 6.5, 10 mM mercaptoethanol, and dialyzed overnight against two 2-liter changes of this buffer in the cold.
Small volumes of dialysate were diluted with 10 mM mercaptoethanol to give a conductivity corresponding to that of 20 mM potassium phosphate, and these were then loaded onto a phosphocellulose column (1.5 cm (diameter) X 40 cm) which had been equilibrated with 20 mM potassium phosphate, pH 6.5, 10 mM mercaptoethanol.
When loading was complete, the column was washed overnight with 5 column volumes of equilibration buffer until base-line absorbance was reached. The column was then eluted with a 600-ml linear gradient of 20 to 200 mM potassium phosphate, pH 6.5, 10 mu mercaptoethanol.
DNA polymerase I elutes as a substantial protein peak at 75 to 150 mM phosphate with an enzymatic activity profile (polymerization) closely corresponding to the protein profile. Electrophoresis of the column fractions in sodium dodecyl sulfate polyacrylamide gels indicated that they were composed of one major protein species with a subunit molecular weight of 109,000, identical in mass with that of DNA polymerase I prepared by other methods (5).
Ammonium sulfate was added to the pooled phosphocellulose column peak fractions (165 ml, 1.2 mg/ml of protein) with stirring in the cold to 85% saturation (15). After standing overnight the precipitate was collected by centrifugation at 15,000 X g for 25 min. This material was stored at -50°C as the pellet pending further purification or use in enzymatic or protein chemical experiments.
The phosphocellulose column yields a preparation of DNA polymerase I which is essentially pure by gel electrophoresis criteria. Concentrated solutions of the enzyme at this stage of purification may have varying amounts of a pale yellow impurity which apparently are carried through the purification procedure as the result of the Polymin P precipitation step. The amount of this impurity varies with the preparation, presumably due to differences in the starting cell pellet. A similar yellow pigment was noted by Jendrisak and Burgess (13) in intermediate stages of their preparation of wheat germ RNA polymerase but was largely removed during ion exchange chromatography.
Final traces of this pigmented material have been removed from the phosphocellulose fractions of DNA polymerase I by chromatography on Bio-Gel P-150 using the chromatography column previously described (5). One-half of the precipitated material from the phosphocellulose column was dissolved in 5 ml of 0.10 M potassium phosphate, pH 7.0, and mixed with 2 ml of reagent grade glycerol. This material was then layered on top of a column (2.5 cm (diameter) x 225 cm) of Bio-Gel P-150 which was equilibrated in 0.10 M potassium phosphate, pH 7.0, 0.10 M ammonium sulfate, 1 mM mercaptoethanol.
The chromatogram was developed in the descending direction with a 50-cm pressure head, and individual fractions were collected and assayed for polymerizing activity, 5' + 3' exonuclease activity, and absorbance at 280 nm. The nuclease assays indicated nucleolytic activity only in fractions corresponding to the main polymerizing peak. This final chromatographic step yields an enzyme  sodium dodecyl sulfate polyacrylamide gel was prepared and loaded with fractions from the different purification stages described in the text. Samples were electrophoresed, stained, destained, and photographed as previously described (1). Migration is from top to bottom in the photograph. Individual tracks are as follows: Tracks 2 and 7, material from Bio-Gel P-150 chromatography; Track 2, crude extract from phage-infected celLs; Track 3, ammonium sulfate cut after Polymin P precipitation and extraction of the pellet; Track 4, column fractions pooled after DEAE-cellulose chromatography; Track 5, material as loaded onto phosphocellulose; Track 6, pooled DNA polymerase I activity peak from the phosphocellulose column elution. polymerase at the various stages of the purification are presented in Table II, and the sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the products is displayed in Fig. 2.
Characterization of the Final Polymerase Preparation-A sample of the enzyme from the Bio-Gel P-150 column fraction was hydrolyzed and the amino acid composition analyzed. The distribution of amino acids in the polypeptide is essentially identical with the distributions observed in previous analyses by other investigators (2,17). These analyses are found in Table III.
The enzyme was successfully cleaved into its two component fragments by the method described by Jacobsen et al. (17). A mixture containing 7 mg of the Bio-Gel P-150 enzyme fraction, 1.15 mg of heavily nicked calf thymus DNA (18), 20 pm01 of potassium chloride, and 315 nmol of potassium phosphate, pH 6.5, in 2.35 ml was incubated for 5 min at 37'C. To this mixture 45 ,ul of a solution containing 100 pg/ml of subtilisin in 0.134 M potassium phosphate, pH 6.5, was added, mixed, and the complete sample was incubated for 30 min at 37°C with occasional agitation.
At the end of the cleavage reaction 60 ~1 of a 24 mg/ml solution of phenylmethylsulfonyl fluoride in dioxane was added and the entire mixture incubated a further 60 min at 0°C. The proteolytic fragments were  freed of DNA using a column (2 cm (diameter) x 1.8 cm) of DEAE Sephadex A-25, diluted, adsorbed to a column (1.5 cm (diameter) x 5 cm) of hydroxylapatite, and eluted with a 350ml gradient of 0.03 M to 0.35 M potassium phosphate, pH 6.3, following the protocol of Jacobsen et al. (17). The column fractions were scanned for ultraviolet absorbance and assayed for DNA polymerase I and 5' + 3' exonuclease activity as described above. The active fractions were pooled, concentrated via adsorption to and desorption from small columns of hydroxylapatite in the buffers used for the gradient chromatography, and then assayed. The yields are shown in Table  IV.
The enzyme was incubated with ColEl closed circular DNA as described under "Experimental Procedures." The ratio of polymerase to DNA circles was calculated as lOO:l, assuming a molecular weight of 4.2 X 10" for ColEl DNA (19) and of 1.09 x lo5 for DNA polymerase (2). No nicking of the ColEl circles was detected at these levels of enzyme to substrate, indicating that the polymerase itself is inactive as an endonuclease under pH and ionic strength conditions used for polymerization reactions and that the levels of any contaminating endonucleolytic activity are undetectable. DISCUSSION We have described a method by which the DNA polymerase specified by XpolA specialized transducing phage may be by guest on March 24, 2020 http://www.jbc.org/ Downloaded from DNA Polymerase I from hpolA Transducing Phage purified rapidly and in good yield. The DNA polymerase preparation which we have isolated is essentially homogeneous in composition and appears to be identical with that produced normally in wild type E. coli B cells (1,17). The enzyme is essentially free of endonuclease contamination and should be useful as a reagent for nick translation labeling of DNA molecules as described by Rigby et al. (20). The large fragment of the enzyme may also be obtained as a by-product of the purification or from cleavage of the whole enzyme and will be of substantial utility in DNA sequencing reactions carried out by the method of Sanger and Coulson (21). In addition, the simplicity of the isolation and the relatively large quantities of the product obtained will simplify further investigation of the structural details of the enzyme and its mechanism of action.