An Exonuclease Specific for Double Stranded Deoxyribonucleic Acid

Abstract A nuclease specific for double stranded DNA has been isolated and partially purified from Hemophilus influenzae. The enzyme has a pH optimum of 8.2 to 8.4 and requires a divalent cation, either Mg++ or Mn++. It is most active on DNA whose size has been reduced by either sonic disruption or limited digestion with pancreatic deoxyribonuclease or spleen acid DNase. The enzyme releases only 5'-mononucleotides and leaves a single stranded segment of DNA which is resistant to further hydrolysis. Furthermore, the enzyme destroys the transforming activity of H. influenzae DNA very slowly. These results indicate that the enzyme is an exonuclease, hydrolyzing from the ends of DNA molecules. In addition, the enzyme also exhibits a DNA-phosphatase activity and releases inorganic phosphate from DNA fragments which terminate in 3'-phosphates, suggesting that this enzyme begins hydrolysis from the 3' end of the DNA strands.

A nuclease specific for double stranded DNA has been isolated and partially purified from Hemophilus influenzae. The enzyme has a pH optimum of 8.2 to 8.4 and requires a divalent cation, either Mg+f or Mn++. It is most active on DNA whose size has been reduced by either sonic disruption or limited digestion with pancreatic deoxyribonuclease or spleen acid DNase. The enzyme releases only S'-mononucleotides and leaves a single stranded segment of DNA which is resistant to further hydrolysis. Furthermore, the enzyme destroys the transforming activity of H. influenzae DNA very slowly. These results indicate that the enzyme is an exonuclease, hydrolyzing from the ends of DNA molecules. In addition, the enzyme also exhibits a DNA-phosphatase activity and releases inorganic phosphate from DNA fragments which terminate in 3'-phosphates, suggesting that this enzyme begins hydrolysis from the 3' end of the DNA strands.
The process of bacterial transformation in H. inJEuen,zae begins with the irreversible uptake of double stranded DNA by competent bacterial cells and ultimately results in the formation of a heteroduplex chromosomal structure in which one strand comes from the donor DNA and the other from the bacterial chromosome (1). Notani and Goodgal (1) have shown that while one intact strand of the transforming DNA is inserted into the bacterial chromosome, the other strand appears to be degraded and the donor atoms reincorporated into the DNA. Furthermore, these authors observed that the size of the unintegrated donor DNA is reduced as a function of time after DNA uptake.
This finding is a possible explanation for the earlier observation that a fraction of the donor-transforming DNA is inactivated after it is taken up by the bacterial cells (2). Recently Steinhart and Herriott (3) have shown that the addition of transforming DNA to a competent preparation of H. inJEuen.zae, whose chromosomes are highly labeled with tritiated thymidine, causes a specific release of radioactive material from the cell. All of the above phenomena suggest that nucleases play an important role in the process of transformation in H. in$luenzae. For example, a reasonable hypothesis which explains the observation that only a single strand of donor DNA is integrated into the chromosome is that an exonuclease degrades one strand of the donor DNA and leaves the other available for integration.
Such a process has been postulated for pneumococcal transformation (4). Furthermore, the DNA-dependent release of radioactive material observed by Steinhart and Herriott (3) might be due to the excision of one strand of the bacterial chromosome by an exonuclease.
Therefore, we undertook a study of the nucleases in H. influenxae and attempted to characterize them and correlate their activity with the process of transformation. This paper reports the isolation and characterization of an exonuclease from H. in&enzae. This enzyme is similar in many respects to exonuclease III of Escherichiu coli (5) and the pneumococcal exonuclease reported by Lacks and Greenberg (6).
The similarity between the H. influenzae enzyme and the pneumococcal exonuclease is especially interesting since not only does bacterial transformation occur in both species, but also the mechanism by which it occurs must be similar since in both species only a single strand of the donor DNA is incorporated into the bacterial chromosome (25).

EXPERIMENTAL PROCEDURE
Preparation of 32P-DNA from H. influenzae-The medium used for labeling cells with 32P was prepared by adding to 40 ml of a 2% (w/v) Neopeptone solution (Difco), 0.5 ml of a 4 mg per ml cysteine-HCl solution, 0.5 ml of 2.5 mg per ml uridine solution, I.75 ml of 20% glucose solution, and 4.0 ml of Earle's salt solution (7) which did not contain glucose or phosphate.
The pH of the resulting solution was adjusted to 7.0 with 1 N NaOH, and 0.05 ml of a stock hemin solution (8), 0.10 ml of a 1 mg per ml nicotinamide adenine dinucleotide solution, and 0.5 mCi of carrier-free inorganic 32P04 were added. The medium was inoculated with about 3 X IO9 freshly grown H. inJluenzae cells suspended in 0.15 M NaCl-0.015 M sodium citrate (Buffer A) and grown for 12 to 16 hours at 37" with gentle shaking.
The labeled cells were collected by centrifugation at 12,000 x Q for 10 min and washed 2 times with 50 ml of Buffer A. They were then lysed by suspending them in 4.0 ml of Buffer A and adding 0.4 ml of a 10% SDS1 solution and incubating them at 45" for 10 min. Solid NaCl (0.5 g) and chloroform-1-octanol solution (9: 1, v/v) (4.4 ml) were added to the resulting viscous suspension.
After shaking vigorously for 1 hour, the phases were separated by cen-

Nucleuses of Helnophilus
Injluenxae Vol. 245,No. 20 trifugation at 3,000 x g for 10 min. The clear upper layer was extracted 2 more times with an equal volume of the chloroforml-octanol solution as before. Finally the upper layer was treated with 20 Hg per ml of pancreatic RNase for 30 min at 37" and extracted twice more with chloroform-octanol.
clear supernatant is the streptomycin-sulfate supernatant fraction.
The DNA in the upper phase was separated from the smaller molecules by passage through a Sephadex G-100 column (2.2 X 30 cm) equilibrated with Buffer A. The fractions containing the DNA were pooled and saved. The final yield of DNA was about 100 pg and contained between 30,000 and 50,000 cpm per pg of DNA.
Solid (NH&SO,(105 g) was added slowly to 290 ml of this fraction with constant stirring to make the final ammoniumsulfate concentration to 65% of saturation. The precipitate was removed by centrifugation and the supernatant dialyzed against three 4-liter batches of 0.02 M Tris, pH 7.6-0.05 M NaCl for a total of 48 hours.
Nuclease Assay Conditions-The assay used for detection of nuclease activity was based on the production of acid-soluble radioactivity from a2P-DNA. The radioactive DNA was diluted in distilled water to a final concentration of 20 mpmoles of DNA nucleotide per ml. When sonicated DN.4 was used, the DNA was sonicated for 1 min in a go-watt ultrasonic disintegrator (Measuring and Scientific Equipment, Limited, London). Denatured DNA was prepared by heating the diluted DNA at 100" for 10 min and quickly cooling the heated DNA solution in an ice bath.
The final assay solution contained 5 mpmoles per ml of DNA nucleotide, 0.1 M Tris-HCl, pH 8.2-0.0025 M MgCls-0.001 M DTT in 0.2 mg per ml of BSA (Armour).
The enzyme was diluted into 0.02 M Tris-HCl, pH 7.6-0.001 M DTT in 0.2 mg per ml of BSA before use. The reaction was started by adding 0.3 ml of the diluted enzyme to 3.2 ml of the assay solution above to give a final volume of 3.5 ml or a multiple thereof. All assays were carried out at 37". The reaction was stopped by adding l-ml aliquots of the reaction mixture to 0.4 ml of a 15 mg per ml herring sperm DNA (Calbiochem) solution in the cold and adding 0.8 ml of a 5% (w/v) trichloracetic acid solution. The mixtures were allowed to sit in the cold for 10 min before the precipitates were removed by centrifugation at 10,000 X g for 10 min. Aliquots (1.5 ml) of the supernatant were added to 0.5 ml of a 4 N NH,~H, 0.01% SDS solution on stainless steel planchets, dried, and counted in a windowless gas flow counter.
The dialyzed ammonium-sulfate supernatant fraction (200 ml) was applied to a column (2.3 X 25 cm) of DEAE-cellulose equilibrated with 0.05 M NaCl-0.02 M Tris, pH 7.6. The column was washed successively with 400 ml of 0.05 M NaCl-0.02 M Tris, pH 7.6; 600 ml of 0.1 M NaCl-0.02 M Tris, pH 7.6; 0.2 M NaCl-0.02 M Tris, pH 7.6, at a flow rate of about 100 ml per hour.
The fractions were assayed for nuclease activity and the tubes with the most activity pooled and frozen at -60".
The nuclease activity eluted as a single peak just before the 0.2 M NaCl-0.02 M Tris buffer wash was started.
Potassium phosphate buffer (2.62 ml), 0.2 M, pH 7.0, was added to 50 ml of the combined DEAE fractions to make the final phosphate concentration 0.01 M. To this solution was added 6.0 ml of a suspension of calcium-phosphate gel Type III Basic (Sigma) containing 0.2-g solids. After allowing the suspension to equilibrate with gentle shaking for 10 min, the calcium-phosphate gel was removed by centrifugation at 12,000 X g for 10 min and resuspended in 16 ml of 0.2 M potassium-phosphate buffer. After 10 min the gel was removed by centrifugation as before and the supernatant dialyzed overnight against 4 liters of 0.02 M Tris-HCl, pH 7.6.
Assay of Biological Activity-H.
iq'luenzae cells were made competent, and the biological activity of enzyme-treated native DNA was tested by bacterial transformation as described by Postel and Goodgal (9).

Preparation of Enzyme-H.
influenzae cells were grown in brain heart infusion broth (Difco) supplemented with hemin and NAD (8) with constant aeration at 37". The cells were harvested in late log phase (A6so = 0.53) by centrifugation.
The culture was checked for contamination by streaking the cells on plates which contained brain heart infusion agar but which were not supplemented with hemin or NAD.
The cells were washed 2 times with 0.02 M Tris (adjusted to pH 7.6 with HCl), 0.01 M NaCl and frozen at -60".
All subsequent operations were carried out at O-4".
Protein Determination-The protein concentration of the bacterial extracts which contained large amounts of nucleic acid (the crude extract and the streptomycin-sulfate supernatant fractions) was determined using the Folin-Ciocalteu reagent as described by Layne (10). The protein concentration of the other fractions was determined by the method of Warburg and Christian (11).
Aspergillus oryzae Preparation-A. oryzae was prepared according to the procedure of Ando (12). The specific activity of the enzyme was 2200 p/lmoles/30 pg of protein.

Materials
An extract was made of the cells by suspending 29 g of thawed cells in 58 ml of 0.02 M Tris-0.05 RI NaCl and passing the suspension two times through a French pressure cell (American Instrument Company, Silver Spring, Maryland) at 14,000 pounds per square inch. The resulting viscous suspension was centrifuged at 27,000 x g for 15 min. The supernatant was decanted and the pellet extracted again with 30 ml of 0.02 M Tris, pH 7.6-0.05 M NaCl and centrifuged as before. The resulting supernatants were combined and centrifuged again at 30,000 x g for 45 min. This is the crude extract.
Enzymes-Crystalline pancreatic deoxyribonuclease and pancreatic ribonuclease were purchased from Worthington.
Partially purified 5'-nucleotidase from snake venom was purchased from Sigma.
Bacterial Strains-H. inJluenzae strain Rd and strains resistant to 2000 pg of streptomycin per ml, 25 pg of novobiocin per ml, or both antibiotics were described by Postel and Goodgal (9).
Antibiotics-Streptomycin sulfate was purchased from Nutritional Biochemicals.
Novobiocin was a generous gift of The Upjohn Company. The crude extract (92 ml) was diluted by adding 184 ml of 0.02 Radioisotopes-Carrier-free 32P-orthopllosphate was purchased M Tris, pH 7.6-0.05 M NaCI, and 45 ml of a fresh solution of from E. R. Squibb and Sons, New Brunswick, New Jersey. streptomycin sulfate (5% w/v) were added dropwise with 3H-DNA isolated from H. influenzae, strain SrNovBb, grown in constant stirring over a 2-hour period.
The resulting precipitate the presence of 3H-thymidine was a generous gift of Mrs. Marwas removed by centrifugation at 12,000 X g for 10 min. The garet Robbins.

Purz',fication and Characterization of Enzyme
Extracts of H. in$uenzae hydrolyze both sonically disrupted and heat-denatured sonically disrupted DNA as assayed by the conversion of DNA into acid-soluble products (Tables I and  II).
When streptomycin sulfate is used to precipitate the nucleic acids in these extracts, the nuclease activity which degrades denatured DNA tends to precipitate with the nucleic acids while the activity towards sonically disrupted DNA remains in the supernatant.
First attempts at separating the enzyme activity which degrades denatured DNA from the nucleic acids in the streptomycin-sulfate precipitate were unsuccessful, so no further purification of this activity was attempted. Table II gives a summary of the purification of the exonuclease from H. infiuenzae found in the streptomycin-sulfate supernatant.
The specific activity of the enzyme eluted from the calcium-phosphate gel indicates an 85-fold purification over the crude extract.
The partially purified enzyme is somewhat 2otI~k--l-l---l-l-l---l~l--l--- unstable at O-4" and was normally stored frozen at -60'. Storage at O-4" for 2 weeks resulted in the loss of about 80% of the enzyme activity.
Repeated freezing and thawing also inactivated the enzyme. Fig. 1 shows pH optimum of the enzyme was about 8.2 to 8.4 under the conditions used in this assay. The enzyme requires a divalent cation, Mg+f or Mn++.
The optimum Mg++ ion concentration for these reaction conditions is about 0.0025 M, and if Mg++ is omitted or EDTA is added, the rat,e of the reaction is less than 2% of the rate at, optimal Mg++ ion concentration.
The optimum magnesium ion concentration is the same if native or denatured DNA is used as the substrate; however, the rate of hydrolysis is greatly reduced.
Manganous ions substitute for magnesium ions with an optimum concentration of around 0.0006 M Mn+f.
Calcium and zinc ions are not effective.
The enzyme is sensitive to increasing ionic strength. For example, increasing the sodium ion concentration in the assay medium to 0.076 tin inhibited the rate by 77%. At a sodium ion concentration of 0.14 M, the inhibition was 91% and at, 0.24 M the inhibition was 98%. Potassium ions also inhibited. Likewise, high levels of commercial tRNA inhibited the enzyme.
Xubstrate Specijicity Fig. 2B shows that the enzyme hydrolyzes sonicated DNA at a much greater rate than it does native or denatured DNA.
In this experiment the rate with native DNA is 4% of that with sonically disrupted DNA, while with denatured DNA the rate is 7.4% of that with sonically disrupted DNA.
In five such experiments with the use of different DNA and enzyme preparations, the rate with native DNA averaged 6.8% of that with sonically disrupted DNA, and with denatured DNA the rate was 8.7% of that of sonically disrupted DNA.
The higher rate obtained with sonically disrupted DNA indicates the possibility that the enzyme is an exonuclease which hydrolyzes at, the end  2. A, effect of concentration of native DNA on hydrolysis by enzyme. The assay was carried out as described under "Methods" at a pH of 3.2 in the presence of MgC12, with an enzyme concentration of 160 units per ml. 0, 80 mpmoles per ml, 0, 40 mflmoles per ml; l , 20 mpmoles per ml of native DNA as nucleotide.
The complete hydrolysis of the DNA would be represented by 2100 cpm. The number of counts observed was always greater than 500 for the 30-min sample and greater than 1000 for longer times. B, assay of enzyme activity on native, sonically disrupted, and heat-denatured DNA.
The assay was carried out as described under "Methods" at a pH of 8.2 in the presence of MgC12, with an enzyme concentration-of 480 units per ml. The sonicallv disruoted DNA and denatured DNA were nrenared as described"under "Methods." 0, sonically disrupted CNA; 17, denatured DNA; l , native DNA. of the DNA chain. This conclusion is supported by the fact that the rates of hydrolysis for sonically disrupted DNA and untreated DNA are proportional to the size of the DNA since the sonically disrupted DNA was approximately 30 times smaller than untreated DNA.
The activity observed with denatured DNA could be due to a contaminating enzyme. The hypothesis that this enzyme is an exonuclease is strengthened by the observation that the rate of hydrolysis of native DNA is proportional to the DNA concentration, but the extent of hydrolysis (the fraction of the added DNA converted to acidsoluble products) remains the same. Under the conditions used above for assaying enzyme activity on native DNA, the number of ends of the DNA chains determines the rate ( Fig. 2A).
Lacks and Greenberg (6) have isolated an exonuclease which begins hydrolysis at single strand nicks in the DNA chain. Since genetic transformation occurs in both Pneumococcus and H.
in&enzae, it is important to determine if the exonuclease from H. injI~enz.ue will also initiate hydrolysis at single strand nicks.
The endonuclease, pancreatic DNase, introduces single strand nicks into DNA.
The number of phosphodiester bonds which this enzyme must break in order to reduce the weight average molecular weight by a factor of 2 has been estimated by Thomas (13) to be 200. Therefore, this enzyme is a useful tool for introducing single strand nicks into DNA.
H. influenzue 32P-DNA was hydrolyzed with 0, 150, 1,500, and 15,000 ring per ml  Fig. 3 and 0.3 Pg of 3H-labeled marker DNA were layered on top of linear 5 to 20% sucrose gradients made up in 1 M NaCl-0.01 M EDTA, pH 7.6. The samples were then centrifuged at 35,000 rpm for 2 hours at 18' in a Spinco SW-39 rotor. Four drop fractions were collected from the bottom of the tube onto cellulose filter paper discs, washed, and counted in a liquid scintillation spectrometer as described before (1). l , 3H-labeled marker DNA; 0, DNase treatment. A, no prior treatment; B, treatment with 1,500 PpLg of DNase; C, treatment with 15,000 Mpg of DNase. of pancreatic DNase at 30" for 30 min. The DNA was reisolated by phenol extraction and tested for its ability to act as a substrate for the H. influenzae enzyme. Fig. 3 shows that the DNA treated with the higher levels of pancreatic DNase is a good substrate for this enzyme. Fig. 4 shows that this DNA sediments more slowly than control DNA in neutral sucrose gradients. Therefore, it seems likely that the increased activity of this enzyme toward DNase-treated DNS was due primarily to double strand breaks.

Nature of Products of Reaction
Acid-soluble Hydrolysis Products--Two lines of evidence indicate that the acid-soluble products released by the H. injZuenzae enzyme were 5'-mononucleotides. Fig. 5 shows that the acidsoluble material produced by the H. in&enzae-catalyzed hydrolysis of sonicated DNA eluted at the same position as 5'mononucleotide standards when chromatographed on Dowex 50-AG by the method of Blattner and Erickson (14). The recovery of the radioactivity from the column was 95%, and the AT/GC ratio of the recovered material was 1.7 which agrees well with the published AT/GC ratio of 1.63 for H. inJluenzue DNA   TABLE   111 Action of snake venom 5'-nucleotidase upon sonically disrupted DNA The acid-soluble hydrolysis products used in this assay were obtained by hydrolyzing sonically disrupted DNA at pH 7.6 in the presence of MgCls as described in the legend to Fig. 6. After adding carrier DNA, one-half of the reaction mixture was stopped by adding 57, trichloracetic acid, the other by adding 0.6 M HC104. After cooling on ice, the precipitates were removed by centrifugation in the cold. The trichloracetic acid was removed by extracting the supernatant 5 times with an equal volume of ether. The HClO, supernatant was neutralized to pH 7 with 2 M KOH, cooled in ice, and the resulting precipitate of KC104 removed by centrifugation.
The snake venom 5'-nucleotidase assay was carried out in a total volume of 0.5 ml containing 0.1 M glycine-NaOH buffer, pH 9-0.01 M MgC12, and the amounts of the acid supernatants and 5'-nucleotidase indicated.
The incubation was for 1 hr at 37", after which the tubes were chilled and the following added in order: 0.7 ml of HSO, 0.2 ml of 0.2 M NaH,PO+ 0.2 ml of 1 N HCl, and 0.4 ml of a 1% acid-washed Norit suspension in HZO. The Norit was removed by centrifugation and the supernatants plated and counted.  5. Separation of acid-soluble products on Dowex 50-AG. 32P-DNA from H. influenzae was sonicated and hydrolyzed by the H. in$uenzae enzyme in the usual manner at pH 7.6 in the presence of MgC& with 1110 units per ml of enzyme. After 1 hour at 37" the reaction was stopped by adding the usual amounts of unlabeled carrier DNA and 0.6 M HClOb in place of 5% trichloracetic acid. This solution was chilled in an ice bath, centrifuged, and the supernatant removed. The pH of the supernatant was adjusted to -3.2 with 2 M KOH in the cold, and the resulting precipitate of KC104 was removed by centrifugation. The neutralized supernatant (0.5 ml) was diluted with 0.5 ml of 0.1 M ammonium for-mate adjusted to pH 3.2 with formic acid, and ohromatographed on a Dowex 50-AG column by the method of Blattner and Erickson (14). Samples (0.7 ml), were collected, dried, and counted in a gas flow counter. The arrozus indicat,e the position of 5'-mononucleotide standards prepared and chromatographed as described before (14). 6. Kinetics of hydrolysis. Son&ted DNA was hydrolyzed at pH 7.6 in the presence of MgCls with 2500 units per ml of enzyme at 37". At l-hour intervals l-ml samples were withdrawn and assayed for the extent of hydrolysis.
At 3 hours. 2 ml were removed, added to 5000 additional units of exonuclease, and the incubation at 37" continued.
This hvdrolvsis mixture was assaved for the extent of hydrolysis at 4 ho&s a& 5 hours after the start of the original incubation. 0, original hydrolysis reaction; 0, results from the sample with added enzyme. (15). The acid-soluble material was also found to be 5'-mononucleotides by paper chromatography.
The second line of evidence that the acid-soluble products were 5'-mononucleotides came from their susceptibility to snake venom 5'-nucleotidase.
This enzyme releases orthophosphate from 5'-mononucleotides but not from 3'-mononucleotides (16). Table III shows that the acid-soluble hydrolysis products are adsorbed to charcoal if no 5'-nucleotidase is added to the reaction mixture.
When the acid-soluble hydrolysis products were treated with this enzyme, an average of 95% of the radioactive phosphate was no longer adsorbed to the charcoal. Control experiments were done to prove the specificity of the 5'-nucleotidase which did not release orthophosphate from 3'-nucleotides ( < 1% of the 5'-nucleotidase activity).
Acid-insoluble Hydrolysis Products-The finding that 5'-nucleotides were released from DNA and that sonic disruption of the DNA greatly increased the rate of hydrolysis suggested that this enzyme was an exonuclease acting at the ends of the DNA chains. The following lines of evidence further substantiated this hypothesis.
If the enzyme-catalyzed reaction was allowed to continue to completion, about 50% of the 32P-DNA was resistant to further hydrolysis. Fig. 6 shows that the addition of more enzyme at this point did not greatly enhance the quantity of material solubilized.
This experiment was repeated at pH 8. 7. CsCl density gradient centrifugation of hydrolysis products.
Native 32P-DNA was hydrolyzed with 1140 units ner ml of H. influenzae exonuclease at pH 8.2in the presence of MnCls as described under "Methods." Aliauots were removed and the degree of hydrolysis determined as usual. At 0 min. 2 hours. and 5 hours after the addition of the enzyme, 2.0-ml al&rots were removed and added to 2.0 ml of 0.4'$& Sarkosyl-0.04 M EDTA, pH 7.6, in the cold. The resulting solution (1.2 ml) was added to 4.52 g of CsCl, -0.2 fig of W-labeled native DNA and 22 ,ug of heatdenatured unlabeled DNA. and sufficient HnO to make a final volume of 5.0 ml. The resulting solution was centrifuged as described by Notani and Goodgal (1) to establish a CsCl gradient. Fractions were collected, washed with 57, trichloracetic acid, and counted as before. The sample (29%) taken at 2 hours was acid soluble; at 5 hours 41% was acid-soluble. q , 3H-labeled marker DNA; 0, enzyme-treated DNA.
A, results at 0 min; B, at 2 hours; C, at 5 hours. The top of the gradient is at the right of the figure. presence of manganous ions and at lower DNA concentrations, and in each case between 40 and 60% of the DNA remained resistant to further hydrolysis.
The reaction did not stop completely, but continued at a much slower rate. This result suggests that the enzyme was behaving similarly to exonucleases which hydrolyze double stranded DNA from one end and leave a single strand fragment which is resistant to further hydrolysis (e.g. exonuclease III of E. co& (5) and the X-induced exonuclease 07)).
CsCl density gradient centrifugation was used to investigate the nature of the large molecules remaining after nuclease digestion. Because of its small size, sonically disrupted DNA does not band sharply in CsCl, so the experiment was done with native DNA.
Native 32P-DNA was hydrolyzed in the presence of manganous ions at pH 8.2 for 5 hours, when 41y0 of the DNA was acid soluble.
Two samples were taken during the course of the 8. Hydrolysis of large molecule products of exonuclease digestion bv a nuclease from A. oruzee. Sonicated 32P-DNA was hydrolyzed-with 810 units per ml ofH. influenzae exonuclease and the extent of hydrolysis determined as usual. At 0 min and 3 hours after addition of the enzyme, 4-ml samples were removed and extracted with an equal volume of phenol saturated with Buffer A. After 3 hours of incubation, 810 units per ml of additional enzyme were added and the incubation continued for 3 more hours when another 4-ml sample was removed and extracted with Buffer A-saturated phenol. After the phenol phases were removed by centrifugation, the aqueous phases were extracted 2 times with an equal volume or ether and dialyzed overnight against Buffer A. Samples of DNA from the dialyzed fraction were treated at 37" with 0.08 pg per ml of the nuclease from A. orqzae prepared according to Ando (12 The results given in Fig. 7 show that as hydrolysis proceeded the remaining DNA banded closer to the position of heat-denatured DNA.
The 32P-DNA peaks decreased in size and became progressively broader.
This was due to the reduction in size of the DNA molecules by hydrolysis.
The DNA hydrolyzed for 5 hours banded at the position of denatured DNA.
An exonuclease isolated from A. oryzae by the method of Ando (12) provided a useful tool for investigating the nature of the large molecule products remaining after exonuclease digestion. This enzyme shows a high specificity for single stranded DNA. The rates with which this enzyme hydrolyzed sonically disrupted DNA and heat-denatured DNA are shown in Fig. 8 9. Comparison of the rates of hydrolysis and the loss in biological activity.
32P-DNA isolated from a strain of H. in$uenzae resistant to 25 pg per ml of novobiocin was hydrolyzed by the exonuclease in the presence of MgClz or MnClz at pH 8.2 as before. Samples were removed at intervals to determine the extent of hydrolysis, and at the same time other samples were removed and diluted into cold Buffer A and frozen. After thawing, the biological activity of these samples was determined as described under "Methods." 0, hydrolysis with Mn++; 0, hydrolysis with Mg++. The top of the figure shows biological activity remaining, and the boltom of the figure shows the hydrolysis of native DNA by the exonuclease. also shows that the large molecule products of the exonuclease digestion are readily hydrolyzed by the Ando endonuclease. Furthermore, the rate with which the A. oryzae enzyme hydrolyzed the products of the exonuclease digestion increased as the extent of hydrolysis increased.
These data confirm the conclusion that the large molecule products remaining after exonuclease digestion are single strands of DNA.
Further evidence that the H. influenzae enzyme is in fact an exonuclease comes from a study of the loss of biological activity with increasing hydrolysis. Richardson,Lehman,and Kornberg (18) have reported that with E. coli exonuclease III after 5% hydrolysis, 50% of the biological activity remains as determined by bacterial transformation, and Lacks and Greenberg (6) have reported that even after extensive digestion (37oj, hydrolysis) with the pneumococcal exonuclease, 15% of the original biological activity remains.
Similarly with the H. in$uenzae enzyme, as shown in Fig. 9, there is relatively little loss in biological activity as the extent of hydrolysis increases. The relatively low levels of hydrolysis obtained are due to the fact that native DNA was used in this experiment.
Sonication destroys almost all of the biological activity (19). These results show that this enzyme is not cleaving the DNA molecule endonucleolytically. 10. DNA-phosphatase activity. 5'-Phosphate-terminated DNA was prepared by hydrolyzing azP-DNA (12 pg per ml in 0.01 M Tris, pH 7.0-0.002 M MgClz-0.1 M NaCI) with 40 mpg per ml of pancreatic DNase for 30 min at 37". The reaction was stopped by shaking the reaction mixture with an equal volume of phenol saturated with Buffer A as described under "Methods." 3'-Phosphate-terminated DNA was prepared by hydrolyzing 3*P-DNA (12 pg per ml in 0.1 M sodium acetate, pH 5.0-0.05 M NaCI-0.01 M EDTA) at 30" with 200 mpg per ml of spleen acid DNase for 30 min before phenol extraction as before. Both DNA samples were finally dialyzed against 0.015 M NaCl-0.0015 M sodium citrate. The total amount of inorganic phosphate released by alkaline phosphatase was determined by incubating the hydrolyzed DNA samples (5.5 pg per ml) with 100 pg per ml of alkaline phosphatase (in 0.01 M Tris, pH 8.0-0.01 M MgClt) at 37". At 60 and 120 min, samples were taken and the hydrolysis reaction stopped by chilling and the addition of EDTA to a final concentration of 0.001 M. Aliquots were then chromatographed on Whatman No. 3MM paper using the solvent system of Markham and Smith (22). The region containing the inorganic phosphate was cut out and counted in-a liquid scintillation Fount&.
The samples of DNA hydrolyzed by pancreatic DNase and spleen acid DNase were treated with the H. injluenzae exonuclease under the usual conditions except no BSA was added. At various times samples of the reaction mixture were precipitated with trichloracetic acid to determine the extent of hydrolysis.
The amount of inorganic nhosphate released was determined by stopping the hydr%lysis-with the addition of EDTA to a final concentration of 0.005 M and chromatographing samples as described above. 0. DNase I (5'-terminated DNA); 6, DNase II (3'-terminated DNA). The ton figure shows hvdrolvsis of DNase I-and II-treated DNA bv H:in&enzae exon&lease. The bottom figure shows the inorgani"c phosphate released by the exonuclease. Arrows indicate inorganic phosphate released by alkaline phosphatase from DNA treat,ed with DNase I and II.
Exonuclease III of E. coli and the pneumococcal exonuclease both show DNA-phosphatase activity (5, 6). These enzymes release inorganic phosphate from DNA fragments which terminate in 3'-phosphomonoesters.
Spleen acid DNase and pancreatic DNase are useful tools for producing 3'-and 5'-phosphate-terminated DNA fragments (20, 21). Fig. 10 shows that the H. infEuenzae exonuclease rapidly releases inorganic phosphate from DNA fragments produced by spleen acid DNase (3'phosphate terminal fragments) but not from pancreatic DNaseproduced fragments (5'9erminal phosphates). There is a very slow linear release of inorganic phosphate from 5'-phosphateterminated fragments which might be a result of a contaminating nucleotidase or phosphatase. The total amount of inorganic phosphate released from 3'-phosphate-terminated DNA is 75 % of the inorganic phosphate released by alkaline phosphatase. This failure to obtain a complete release of inorganic phosphate might be due to the presence of either internal phosphomonoesters or terminal 5'-phosphomonoesters. DISCUSSION The finding that the H. injluenzae enzyme hydrolyzes sonically disrupted DNA at a much greater rate than it does intact DNA first suggested that the ends of the DNA molecule were the initial site of hydrolysis and that this enzyme was an exonuclease. This assumption was strengthened by the finding that the acid-soluble products released are 5'-mononucleotides.
The fact that the enzyme rapidly hydrolyzes about 50% of the DNA and that further hydrolysis occurs at only about 5% of the initial rate was explained by the finding that the acid-insoluble product remaining is single stranded DNA.
These results show the enzyme is in fact an exonuclease and hydrolyzes DNA molecules in a stepwise manner from the ends of the molecule until single strand molecules are formed.
The finding that this enzyme possesses a DNA-phosphatase activity specific for DNA fragments terminating in 3'-phosphates further suggests that the initial site of hydrolysis occurs at the 3' ends of the DNA chains. If a 3'phosphate is present, it is released as inorganic phosphate before 5'-mononucleotides are released. As Lacks has pointed out in Lacks and Greenberg (6), the data on the inactivation of the transforming activity by this enzyme should be viewed with caution.
It is possible that the enzyme is selectively hydrolyzing small DNA molecules which do not transform despite the fact an excess of enzyme is used in these experiments. Alternatively, the DNA molecules which do transform may be resistant to hydrolyzing by the exonuclease.
The mechanism by which this enzyme hydrolyzes DNA is similar to that of exonuclease III of E. co& (5), an exonuclease from Pneumococcus (6), and an exonuclease from Bacillus subtilis (23). It is interesting to note that bacterial transformation is a well known phenomenon in H. in$uenme, Pneumococcus, and B. subtilis, and there has recently appeared a report that transformation also occurs in E. coli (24). While it is possible that this coincidence has nothing to do with the process of transformation per se, it is interesting to see how such an enzyme might play a role in this process.
In both H. inJEuenxae and pneumococcal transformation, only one strand of the donor-transforming DNA is incorporated into the bacterial chromosome (1,25). In Pneumococcus this is explained by the finding that the donor DNA is hydrolyzed to mononucleotides and single stranded DNA (4) soon after the DNA is bound to the competent cells. The situation is different in H. in$uenxae in that no single stranded DNA can be found during transformation despite the fact only a single strand is ultimately incorporated into the chromosome (1). However, it could be that integration occurs so fast in this species that no pool of single stranded material can accumulate. An enzyme such as Methods in enzymology, Vol. III, Academic Press, New York, this exonuclease is a likely candidate for the production of single 1957, p. 447. stranded material. These enzymes could, of course, play another 11. WARBURG, O., AND CHRISTIAN,W.,Biochem. Z.,310,384 role in the transformation process. For examPl% they could 12. A:??$ Biochim Biophys Acta 114 158 (1966) excise one strand of the bacterial chromosome to prepare a region 13. THO&S,'~.