Substrate Specificity and Adenosine Triphosphatase Activity of the ATP-Dependent Deoxyribonuclease, of Bacillus subtilis

Eur J. Biochem 114,493-499 (1981) FEBS 1981

Substrate Specificity and Adenosine Triphosphatase Activity of the ATP-Dependent Deoxyribonuclease of Bacillus subtilis

Janine DOLY, Daniele LE ROSCOUET, and Constantine ANAGNOSTOPOULOS

Centre de Genetique Moleculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette

(Received May 19/Novem ber 2 I , 1980)

Studies on the specificity of the ATP-dependent DNase of Bacillus subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single- stranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The double- stranded DNA can be completely hydrolysed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3' or the 5' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent K, for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.

The ATP-dependent DNase (exonuclease V), which also possesses DNA-dependent ATPase activity, has attracted much interest on account of its possible involvement in genetic recombination and DNA repair and of its particular mechanism of action. These studies have so far concerned the enzymes found in many bacterial species, as reviewed recentIy by Whitehead et aI. [I]. Although they all share some general features in common they differ individually at certain points, especially as regards substrate specificity, mode of action on DNA, limit products or levels of ATPase activity.

In previous papers we reported studies on the purification, subunit structure and some properties of ATP-dependent DNase of Bacillus subtilis and on a mutant devoid of this activity [2,3]. The enzyme was obtained in an electrophoretically homogeneous state. The molecular weight estimated was 308000 and the molecule was found to be composed of five nonidentical subunits with the following Mr: 81 000, 70000, 62000, 52500 and 42500. The pH optimum and the optimal concentrations for Mg2+ and ATP were determined [3]. Studying in more detail the action of the pure enzyme in vitro should lead to some insight into its eventual role in genetic recombination. An endonucleolytic activity on circular DNA substrates suggests action on the recipient chromosome, probably prior to the formation of the donor- recipient complex (in transformation or transduction). A low activity was previously observed on heat-denatured DNA 131. If this was confirmed with pure single-stranded linear DNA segments it could imply the elimination of single- stranded stretches from either the donor or the recipient,

Abbreviation. SV40, simian virus 40. Enzymes. ATP-dependent deoxyribonuclease or exodeoxyribo-

nuclease V (EC 3.1.11.5); pancreatic DNase or deoxyribonuclease I (EC 3.1.21.1); deoxyribonuclease I1 or acid DNase(EC 3.1.22.1); alkaline phosphatase (EC 3.1.3.1); S1 nuclease or endonuclease S1 (Aspergillus) (EC 3.1.30.1).

____

in a later stage of the recombination process. On the other hand, studies on the course of the duplex linear DNA degradation and its limit product(s) could reveal the formation of significant transient intermediates. Finally in order to achieve a better understanding of the role of ATP in the DNase activity of the enzyme, determination of certain para- meters of its ATPase activity was necessary.

The present paper provides evidence that the pure enzyme of B. subtilis behaves in vitro as an exonuclease detaching mononucleotides one by one from only double-stranded linear DNA while concomitantly displaying a very high ATPase activity.

MATERIALS AND METHODS

Chemicals

[k~'ethyl-~H]Thymidine and [32P]phosphoric acid were supplied by C. E.A. (Saclay, France). [8-3H]ATP and [Y-~'P]- ATP were purchased from the Radiochemical Center (Amer- sham, Great Britain). All other compounds were obtained from sources indicated previously [3].

Enzymes

Electrophoretically pure ATP-dependent DNase of Ba- cillus subtilis 168 was used throughout this work [3]. Its specific activity was about 200-250 units . mg protein-'. There was no difference in the specific activity of the preparations obtained with or without inhibitors of proteases (3 mM phenylmethylsulfonyl fluoride and 2 mM diisopropyl fluoro- phosphate) during the extraction and purification.

Pancreatic DNase (2600 units . mg-') and alkaline phosphatase of Escherichia coli (40 units . mg-') were purchased from Worthington, S1 nuclease of Aspergillus oryzae (1.5 x lo5 units mg-') from Miles and acid DNase of hog spleen ( E 20000 units . mg-') was a gift of Dr G. Bernardi.

494

DNA Substrates

3H-labelled DNAs from B. subtilis and phage T7 were prepared as described by Doly and Anagnostopoulos [3]. Linear single-stranded DNA was obtained from native [3H]- DNA of B. suhtilis by isolation of the complementary strands after alkali denaturation by chromatography on a methylated albumin-kieselguhr column following the technique of Colli and Oishi [4]. Two peaks were obtained, corresponding to the heavy and light strands. The fractions of each peak in which complete hydrolysis by S1 nuclease could occur were pooled and these preparations were used as substrates.

"P-labelled DNA of B. suhti1i.r and phage T7 was extracted and purified as previously described [3] from cultures and lysates obtained in a low-phosphate medium: (0.12 M Tris, 80 mM NaCI, 20 mM KCI, 20 mM NH4CI, 3 mM Na2S04, 1 mM MgC12, 0.2 mM CaCI2, 0.5% glucose, 0.57; Norit- treated Difco casamino acids, 10 pM KHzPO,; pH 7.5) with 20 mCi of a [32P]phosphoric acid solution/l. The specific activity of these DNAs was in the range 1 - 5 x lo4 counts . min-' . nmol ' (as nucleotide residues).

[3 HIDNA of SV40 form I (supercoiled double-stranded circular molecule, 8.5 x 104 counts . min-' . pg-') was a generous gift of Dr Chambon. Form I1 was obtained by intro- duction of nicks into form I with pancreatic DNase according to a modification of the procedure of Goulian et al. [5]. The reaction mixture contained the following, in a 0.10-ml volume : 3 pg of form I, 10 mM Tris/HCl (pH 7 3 , 2 mM MgCl2, 20 pg pancreatic DNase. Incubation was carried out for 90 s at 25°C and the reaction was stopped by heating for 10 min at 67 -C.

[3H]DNA of phage fd (single-standed circular molecules) was extracted from phage propagated in E. coli strain 993 (originated from Dr S. Linn's laboratory). Cells were grown at 37'C in a synthetic medium: 40 mM Tris, 2.5 mM Na2S04, 37 mM NH4CI, 25 mM KCI, 2.5 mM MgC12,0.2 mM Na2HP04, 0.5 ';() glucose, 0.30 casamino acids (Difco), 0.01 :~i1 thiamin, pH 7.0. When the culture reached about 5 x lo7 cells . ml-' it was infected with phage fd (provided by Dr Saucier), at a multiplicity of 10. Infection was followed by the addition of 12.5 mM CaC12, 5 mCi [3H]thymine (specific activity 40 Ci . ininol- ' ) and 50 mg deoxyadenosine. After 4- 8 h the culture was centrifuged, phage isolated according to Bujard [6] and phage DNA extracted and purified following the procedure of Marvin and Schaller [7]. Its specific activity was of 3.0 x lo4 counts . min- . pg-'. Linear segments with 3'-OH and 5'-P termini were obtained from this DNA after mild hydrolysis by pancreatic DNase in the following reaction mixture (0.5 ml): 20 mM Tris/HCl pH 8.2, 2 mM MgC12,4 mM 2-mercaptoethanol, 50 pg fd [3H]DNA, 100 pg bovine serum albumin and 12 ng pancreatic DNase. After a 10-min incubation at 37 C, the mixture was heated for 10 min at 90°C and dialysed overnight against 20 mM Tris/HCl buffer, pH 8.2.

3'-OH/5'-P and 3'-P/S'-OH-terminated linear double- stranded ColEl ["PJDNAs were obtained by treatment of the circular plasmid with the appropriate DNases. The plasmid was prepared according to Clewell and Helinski [8] from E. coli strain JC412 (CoE1) grown in a low-phosphate medium 191. 5'-32P-labelled termini were obtained by pancreatic DNase action in the following mixture (0.50 ml): 20 mM glycine/NaOH buffer pH 9.2, 5 mM 2-mercaptoethanol, 3 mM MgC12, 100 nmol plasmid ["PIDNA (1.8 x 104 counts . min-' . nmol-') 100 pg serum albumin, 12 ng pancreatic DNase. After a 10-min incubation at 37°C

enzymatic action was stopped by heating for 10 min at 90 'C. The 3'-32P-terminated segments were produced after hydrolysis with hog spleen acid DNase in 0.50 ml of the following mixture: 10 mM acetate buffer pH 5.0, 2 mM EDTA, 2.5 mM 2-mercaptoethanol, 100 nmol ColEl ["PI- DNA, 100 pg serum albumin, 10 ng acid DNase. Other conditions of treatment were as for the 5'-32P-terminated segments.

The linear double-stranded [5'-32P]DNA of SV40 (a Hind111 fragment) was a gift of Dr Contreras.

Assuy of ATP-Dependent DNase Activity (Exonuclecise) Unless otherwise stated the standard assay mixture

(0.5 ml) was of the following composition: 50 mM MgClz, 2.5 mM 2-mercaptoethanol, 0.10 M glycine/NaOH buffer pH 9.2, 75.0 pM ATP, 20 pM labelled DNA substrate (as nucleotide residues), 0.20 mg serum albumin, and 2 units enzyme. Control assays (without ATP and without enzyme) were included in all experiments. After 30 min at 40"C, the reaction was stopped at 0 'C by adding 0.20 ml calf thymus DNA solution (2 mg/ml) and 0.15 ml trichloroacetic acid (50%). After 15 min at 0'-C the mixtnres were centrifuged at l0000xg for I5 min and radioactivity determined by scintillation counting in 0.50 ml of the supernatant. One unit is defined as the amount of enzyme which degrades 1 nmol of DNA nucleotide to acid-soluble material under the above conditions.

Assuy of DNA-Dependent ATPuse Activitj'

The composition of the assay mixtures was the same as for the nuclease activity with the difference that 5 pCi of [8-3H]ATP (75 pM) were added per assay. Control assays without DNA and witout enzyme were also run. Incubation was carried out for 30 rnin at 40°C and degradation of ATP was evaluated (20-p1 samples) by measuring the amount of ATP produced as described [3]. A unit of ATPase is defined as the amount of enzyme which hydrolyses 1 nmol ATP in 30 min in the presence of DNA.

Determinution of the Limit Products of DNA Hydrolysis

The reaction mixture (1 ml) contained: 10 mM glycinei NaOH buffer pH 9.2, 5.0 mM MgC12, 2.0 mM 2-mercaptoethanol, 50 nmol ATP, 0.2 mg serum albumin, 50 nmol [32P]DNA of T7 or B. subtilis (5-7x lo4 counts . min-' . nmol-') and 20 units of B. suhtilis ATP-dependent DNase. After 30 min at 40 "C 20 units more of enzyme and 100 nmol of ATP were added and incubation was continued for 60 min. The reaction was stopped by heating for 15 min at 65 'C and the products of DNA hydrolysis were fractionated according to Tomlinson and Tener [ 101.

In the case of the T7 DNA a limit digest by pancreatic DNase was used as marker. The reaction mixture, containing 10 mM Tris/HCl buffer pH 7.4, 15 mM magnesium acetate, 17.5 nmol T7 [3H]DNA (4 x lo4 counts . min-' . nmol-I), 4 mg salmon sperm DNA and 30 pg pancreatic DNase, was incubated at 37 "C for 30 min. The DNase was then inactivated by heating at 100°C for 10 min. A sample of this mixture (0.33 ml) was mixed with the ATP-dependent DNase hydro- lysate (1.0 ml) of the T7 [32P]DNA. For the B. subtilis DNA no such marker was added.

A DEAE-cellulose column (1.5 x 16 cm) was equilibrated with 0.02 M Tris/HCl pH 7.6 buffer containing 7 M urea. The samples previously adjusted to the composition of this

495

buffer were applied on the column and elution was carried out by a 0-0.30 M linear gradient of NaCl in the same buffer, with a flow rate of 1 ml . min-’.

Fractions (10 ml) were collected and radioactivity was measured in 0.5-ml aliquots as described in Fig. 1. At the end of the gradient the column was washed with 100ml of 2 M NaCl in the above buffer to retrieve any residual radioactivity ; in most cases only insignificant amounts of labelled material were found in this washing. More than 95% of the radioactivity added to the column was eluted during the chromatography.

Identfication of P , Released in the ATPuse Reaction

Inorganic phosphate was characterized in the digests by chromatography on Dowex 1x8 columns. The reaction mixture (0.6 ml) contained the following: 67 mM glycine/ NaOH buffer pH 9.2, 33 mM MgC12, 8.3 mM 2-mercaptoethanol, 40 pg serum albumin, 60 pg B. subtilis unlabelled DNA, 67 pM [Y-~’P]ATP, 10 units of ATP-dependent DNase. After a 60-min incubation at 40°C, 0.40 mg serum albumin and 1 .Om1 10 trichloroacetic acidwere added. After a 10-min incubation at 0 ’C the supernatant was treated twice with an equal volume of a 20% suspension of acid-washed Norit. After centrifugation the supernatants were extracted with ether. Unlabelled P, (50 lmol) was added as a marker to the diluted supernatants. The samples were absorbed on columns (1 x 5 cm) of Dowex 1x8 (CI- form) previously washed with 1 mM HCI (50 ml). Elution was carried out with 5 mM HCI and 2.5-ml fractions were collected. The radioactivity of each fraction was measured and the P, content determined colorimetrically [I 11.

Determinution of the Number of Phosphodiester B o n h Split in DNA

The number of phosphodiester bonds cleaved by the enzyme during its action on B. subailis [32P]DNA was determined by a modification of the method of Kelly and Smith [12]. Aliquots (10 pl) of enzymatic digests (see legend of Fig. 9) were incubated with 0.2 unit alkaline phosphatase for 30 min at 37 “C. Aliquots ( 5 pl) of these mixtures were applied on 1-cm squares of poly(ethy1eneimine)-cellulose for measuring total radioactivity. Other 5-yl samples were streaked on strips of poly(ethy1eneimine)-cellulose (1 x 5 cm) at 1 cm from the bottom edge. These strips were also bearing a band (1-cm width) of Norit added as 20% suspension at 2 cm from the bottom edge. The strips were chromatographed in 1 M HCI. 32P,-liberated by the phosphatase is not retained by Norit and it is found in the upper 1.5 cm of the strip. This part was cut and its radioactivity was measured. The phosphodiester bonds split were calculated as the ratio of (32P in Pi/total 32P) x mol DNA nucleotides.

RESULTS

Substrate Specificity

All double-stranded substrates are readily degraded regardless of their source (Table 1). 5‘-P/3’-OH and 3’-P/5‘OH- terminated ColEl DNA fragments were hydrolyzed at the same rate. In all cases the reaction proceeds to total hydrolysis if the incubation time is increased and more enzyme is added. Since the enzyme does not degrade single- stranded DNA under the conditions of the experiments (see below) these results indicate that it can attack each strand from either end, regardless of whether a terminal orthophos-

Table 1. ATP-dependent DNase activity on double and single-stranded linear DNA from various sources The assays contained 10 nmol of substrate, except for SV40 DNA (= 6.7 nmol) and 2 units of enzyme. All other conditions were those of the standard assay

Substrate Acid-soluble nucleotides formed

Double-stranded : B. subtilis [3H]DNA T7 [32P]DNA CoEl [5’-32P]DNA (3’-OH) ColEl [3‘-32P]DNA (5’-OH] SV40 [5’-32P]DNA (3’-OH)

B. subtilis heavy-strand [3H]DNA B. subtilis light strand [”]DNA fd 5’-P/3’-OH 3H-labelled fragments

Single-stranded :

nmol

4.0 2.7 1.35 1 .50 1.90

0 0 0

( X total)

(40.0) (27.0) (13.5) (15.0) (28.5)

phate residue is or is not present at the site and that reaction rates from both ends are very similar if not identical.

Single-stranded linear DNAs, in contrast, are not attacked by the Bacillus subtilis DNase. This is true for the heavy and light strands of B. subtilis DNA isolated by chromatography on a methylated albumin/kieselguhr column as well as for the 5’-P/3’-OH-terminated DNA of the single-stranded phage fd. NO release of acid-soluble radioactive material was observed from these substrates even after prolonging incubation up to 90 min or adding more enzyme (3 units) and more ATP (75 pmol). These substrates were totally hydrolysed by S1 nuclease. This lack of activity of the B. subtilis DNase on pure single-stranded DNA could be ascribed to the particular conformation of the substrate molecule. In such bush-like structures threading of the enzyme at the free ends and progress along the strand may be hindered. However, such folding of the chain must also occur in the solutions of denatured unfractionated DNA which is hydrolysed slowly to some extent by this enzyme [3]. Moreover linear fragments of fd DNA are digested by the Escherichia coli and H . in- Jluenzae ATP-dependent DNases [13,14]. It seems more likely that the B. subtilis DNase of our preparations is unable to act upon single-stranded DNA. The observed low activity on unfractionated denatured DNA may therefore reflect the presence of partially annealed (or incompletely denatured) DNA segments, possessing double-stranded streches at one end.

Three circular DNA substrates were tested : supercoiled SV40 DNA (form I); double-stranded circular with single strand breaks (SV40 form 11) and single-stranded circular DNA of phage fd. In the same conditions as for the linear DNAs the enzyme did not release any acid-soluble material from them even after prolonged incubation. Shemyakin et al. [I51 found that supercoiled and circular relaxed ColEl DNA were not hydrolysed by purified ATP-dependent DNase of B. subtilis prepared by a procedure different from ours.

The enzyme, however, might possess endonuclease activity and form acid-insoluble segments, which in the case of the two forms of SV40 could have long single-stranded tails. To test this possibility we studied the sedimentation of these substrates after exposure to the enzyme. Incubation mixtures were centrifuged in neutral sucrose gradient. The effect of the enzyme on the three circular substrates was compared to that on B. subtilis linear double-stranded DNA (Fig. 1 - 4).

A C

0 10 20 30 400 10 20 30 400 10 20 30 40 Fract ion number

Fig. 1. Action oftlie A 7P-tlepenm'L.nt DNusr on B. subtilis native D N A . Reaction mixtures (0.10 ml) total volume contained 3.2 nmol B . suh t ih [3H]DNA (specific activity 1 .56 Y 10" counts min- ' nmol DNA-'). ATP was added in tubes A and B and enzyme ( 3 units) in tubes B and C. No serum albumin was added. Mixtures wcrc incubated for 60 min at 40 'C and reaction was stopped by the addition of 33 mM EDTA (pH 8.0). Each mixture was layered at the top of a linear ncutral sucrose gradient (5-20% in a solution of 75 mM NaCI, 7.5 mM sodium citrate) and centrifuged at 200000 x g with rotor SW 50L for 3 h a! 4 C. Forty fractions were collected through the bottom of the tubes and their radioactivity was measured after adding 5 ml of scintillation fluid (5.5 9 PPO. 0.10 g POPOP, 333 ml Triton X-100 SL, 667 ml toluene)

Fyaction number

Fig 2 Action of ihr A I P-depniIen{ D N u x on supercoiled D N A /SV40 form I ) Reaction mixture\ Lontained 0 60 pg SV40 form I 13H]DNA (8 5 x lo4 count5 min pg ' ) (0- --0) With enzyme ( 3 units), (0 --0) no en7ymc ATP was present in both tubes The experiment wdb performed a i deicribed i n Fig 1

As expected the native DNA of B. subtilis is considerably degraded by the e n p m e to acid-soluble material after 60 min at 40°C in the conditions of these experiments and in the presence of ATP (Fig. 1 €3). A second peak of acid-insoluble and slowly sedimenting DNA also appears. This may cor- respond to DNA segments attacked by the enzyme but incompletely degraded a t the time the reaction was stopped. In the absence of ATP (C). as in the absence of enzyme (A), the sedimentation profile shows only the presence of the intact double-stranded material. indicating again the absolute requirement of ATP f o r the reaction.

The supercoiled DNA (SV40 form I) is not attacked by the B. .sarhtilis ATP-dependent DNase (Fig. 2). There is no change in the sedimentation velocity, no decrease in the quantity of the substratc added and no appearance of acid-soluble 3H-labelled compounds in the presence of enzyme (with or without ATP).

Treatment of the supercoiled DNA of SV40 (form I) with pancreatic DNase (as described in Materials and Methods) introduces _S'-P13'-0I-I cuts in one strand. The nicked substrate is SV40 form 11. This conversion is not complete. There was

5 15 25 35 Fraction number

Fig.3. Action of' the ATP-dependent DNu.re on u nicked circular D N A (SV4flform I I ) . The reaction mixture contained 0.60 pg SV40 [3H]DNA (8.5 x I e c o u n t s min- ' pg-') treated with pancreatic DNase asdescribed in Materials and Methods. This preparation was composed of about 40Ya of form I1 and 60 7; of form I . (0 ~- ~--O) No enzyme (w- - -0) 3 units of ATP-dependent DNase. ATP was present in both tubes. The experiment was performed as described in Fig. I .

about 40% of form I1 in the preparation we used which explains the presence of the two peaks in the sucrose gradients (corresponding to intact form I and form 11, Fig.3). Form I1 also is not attacked by the B. suhtilis DNase as shown by comparing the sucrose gradient centrifugation profiles of the assays with and without enzyme (Fig. 3).

Similar negative results were obtained with the circular single-stranded DNA of phage fd (Fig.4). This substrate is also not degraded or otherwise altered by B. suhtilis ATP- dependent DNase preparations.

The above results with circular DNA substrates are consistent with the hypothesis that this DNase preparation does not display any endonuclease activity.

Double-stranded circular DNAs, whether supercoiled or nicked, are also not attacked by the purified. E. coli [13,16] or H. influenzae [14] ATP-dependent DNases. Circular-single- stranded DNA of phage fd is, however, hydrolysed by these enzymes [13,14,17].

497

I I 6 1

0 0 5 10 15 20 25 30 35

Fraction number Fig.4. Action qf the ATP-dependent DNase on the single-stranded circular DNA of phage f d . The reaction mixtures contained 2.5 pg of fd [3H]DNA ( 3 . 0 ~ lo4 counts min-' pg-I). ( 0 0 ) No enzyme; (*--+) 3 units of ATP-dependent DNase. ATP was present in both tubes. The experiment was performed as described in Fig. 1

Limit Product of' DNuse Action [32P]DNA of phage T7 was totally hydrolysed by the

enzyme and the acid-soluble nucleotides released were characterized by chromatography on DEAE-cellulose in the presence of 7M urea. A digest of T7 [3H]DNA by pancreatic DNase was included as internal marker. Only mononucleotides are produced by the action of the ATP-dependent DNase of B. subtilis (Fig. 5) . Similarly when the substrate was [32P]DNA of B. suhtilis a single peak of liberated nucleotides was observed corresponding to the position of AMP (Fig. 6). Partial hydrolysates of both DNAs (from T7 and B. subtilis) obtained by shorter time exposure to the enzyme and treated in the same way again contained mononucleotides as sole components in the acid-soluble fraction. The data with the T7 DNA are included in Fig. 5. These studies on the limit product lead to the conclusion that the B. subtilis ATP-dependent DNase detaches mononucleotides one by one from the double-stranded DNA segments till total hydrolysis. This finding is rather surprising since all similar DNases of other microorganisms studied produce as limit products few mononucleotides and mostly short-chain oligonucleotides with an average of 3-6 units [13,18-221.

ATPuse Activity

Like all ATP-dependent DNases, the B. subtilis enzyme has an absolute requirement for ATP in the exonuclease reaction. There is a very sharp optimum of ATP concentration (70 pM) [3].The only other nucleotide that can partially replace ATP is dATP. On this point the B. subtilis DNase resembles that of Micrococcus luteus [23] and differs from those of E. coli [24], H . injluenzue [25] and B. luterosporus [26] which show significant activity in the presence of other nucleotide triphosphates. ATP is degraded during the reaction and its hydrolysis is dependent on the presence of DNA. Since the studies reported above on the substrate specificity have shown that the pure B. subtilis enzyme acts as an exonuclease on double-stranded DNA alone giving mononucleotides as sole products it became all the more interesting to investigate its ATPase activity in the hope of elucidating the relationship between the two functions. Data are presented concerning the following aspects of the ATPase activity of the purified enzyme: (a) the products of ATP hydrolysis, (b) the determination of the affinity of the enzyme for ATP, (c) the stoichiometry of ATP molecules consumed in relation to DNA phosphodiester bonds split.

/

0 10 20 30 40 50 60 70 80 '

0.3

0.2 g - 0 m

0.1 2

0 1

Fraction number

Fig.5. Limit product obtained from T7 D N A by ATP-dependent DNase action as compared to a pancreatic DNase digest of this substrate. Chromatography of the hydrolysates on DEAE-cellulose in the presence of 7 M urea. The radioactivity figures are those measured in the 0.5-ml samples of each fraction. (0) Hydrolysis of T7 [32P]DNA by ATP-dependent DNase for 90 min at 40°C; (0) hydrolysis of T7 [3H]DNA by pancreatic DNase for 90 min at 37 "C. The straight line indicates the NaCl concentration in the elution gradient. Shorter time exposures of the T7 DNA to the ATP-dependent DNase: (A----A) 15 min; (G---O) 30 min

6 I

0 10 20 30 40 50 60 70 80 90 100

Fract ion number

Fig.6. Limit product of hydrolysis of B. subtilis ("PjDNA by the ATP- dependent DNase. Conditions of the reaction and the chromatography of the product as for Fig. 5. ( L O ) Hydrolysis of B. subtilis DNA for 90 min at 40 "C; (O----o) position of dAMP (A260) chromatographed separately in the same conditions. Straight line = NaCl concentration

In our previous work, as well as in most of the experiments of the present paper, ATP hydrolysis was estimated by the amount of ADP liberated. It was necessary to assess whether the second product, inorganic orthophosphate (Pi), is also released in free form and whether its amount is equimolar to that of ADP.

[y-32P]ATP was incubated with 10 units of enzyme for 60 min at 40 "C in the presence and absence of DNA. The assays were treated and chromatographed as described in Materials and Methods. As shown in Fig.7 the 32P radio-

:'o 30 40 50

11.5

1: 60

Fraction number

Fig. I. Ident~fic~ariotr o/ P, wleu.red f rom A T P in the course of the DNase reaction. Chromatography on Dowex 1x8 of the acid-soluble material non-absorbable by Norit which is liberated from ATP during the DNase reaction. (0) "P radioactivity: (0) P,

activity peak of the eluted material coincides with that of the colorimetrically determined Pi. In another series of experiments the enzyme reaction mixtures (in the same conditions as above) contained both [.y3*P]ATP and [8-3H]ATP. The amounts of ADP and Pi were determined in aliquots of these mixtures. ADP was estimated, as usual, after poly- (ethy1eneimine)-cellulose chromatography and Pi as for Fig. 7. The molar amount of Pi liberated was equivalent to that of ADP (data not shown). These results were obtained in the presence of double-stranded linear DNA. In its absence, as previously observed, there is no hydrolysis of ATP whatever the time of incubation. This-DNA-dependent ATPase therefore splits ATP. liberating ADP and Pi in equimolar amounts. Both products are also released from ATP by the enzymes of E. c d i [13], H . infhrenzue [24] and M. luteus [23].

The apparent K,,, of the B. suhtilis enzyme for ATP (in the ATPase reaction) was measured with concentrations of the nucleotide ranging over 9- 115 pM in the presence of excess B. suhtilis DNA. A Lineweaver-Burk plot of the results is given in Fig.8. From the points that fall on the straight line it was calculated that the apparent K,,, of the B. subtilis enzyme in our preparations is 0.15 mM. This is very close to the value reported for the purified H. influenzur DNase (0.18 mM) [24]. However at concentrations higher than 60 pM (86.5 and 115.0 pM) ATP has an activating effect on its own hydrolysis by the B. .suhrilis DNase (Fig.8). It must be recalled that such ATP concentrations are inhibitory for the DNase activity of this enzyme [3]. The significance of this activation is not understood.

We studied the stoichiometry of ATP consumption in the DNase reaction. I n these experiments the reaction mixture contained [8-3H]ATP and B. suhtilis [32P]DNA. The number of [3H]ADP molecules released and that of the DNA phosphodiester bonds split were determined as a function of time of incubation at 40 C. Experimental details are given in Materials and Methods and in the legend of Fig.9. From the data of Fig.9 it was calculated that the ratio of ADP molecules released to phosphodiester bond breaks is 3.2. Ohi and Sueoka [27] postulated that the B. suhtilis DNase cleaves 30 molecules of ATP per DNA phosphodiester bond broken. This value was, however. calculated on the assumption (inferred from data with the E. c d i and H. influenzue enzymes) that the average length of the acid-soluble oligonucleotides released from DNA is 6 nucleotides. In fact the data of these authors (from single time points) show a ratio of 4.5 for Pi released

I

1 - " I - 21 q /"

7

_IL1-,1 I

0 20 40 60 80 100 120 l/[ATP] (mM ')

-2 Fig 8 Appaient K, for A T P 111 thc DNA-i1epentLnt ATPuw reaction The reaction mixture (0 15 ml) contained 5 pg B w h f h DNA, 1 units of ATP-dependent DNase, and 1 3 - 17 2 nmol [X-'H]ATP (2 4 x 10' counts mm ' nmol I ) After a 30 mm incubation at 40 C the amount of ATP hydrolysed to ADP was determined by ChrOmdtOgrdphy on poly(ethy1eneimine)-cellulose The figure shows d Lineweaver-Burk plot of the results z IS measured as the amount of ATP hydrolysed during the 30-min rcaction

6'0r----11'5 5.0

I /O' I

Time (min)

Fig.9. A T P consumption during the DNcrsr reuc rion. The reaction mixtures (0.25 ml) are described in Materials and Methods except that they contained: 12.5 nmol of [8-3H]ATP ( 8 . 0 ~ lo4 counts min- ' nmol-'); 3.0 nmol of B. subtilis ["PIDNA (2.0 x Id counts min ' . nmol-') and 3 units ofATP-dependent DNase. Incubation uas carried out at 40 C. At intervals 30 pI samples were pipeted into tubes held on ice containing 5 pl of 0.1 M EDTA solution pH 8.0. Samples (10 pl) of these mixtures were taken out for measuring the amount of ATP convcrted to ADP (0) and the number of DNA phosphodiester bonds split (0) (see Materials and Methods). The values are those calculated for the total volume of the incubation mixtures

from ATP to nucleotides released from DNA. The value of 3 molecules of ATP per phosphodiester bond split which we found in our experiments was also reported for the enzymes of M . luteus [23], M . smegmutis [I81 and B. luterosporus [25]. The E. coli and H . influenzue DNases hydrolyse much more ATP: 23 [I31 and 31 -39 [24] molecules, respectively.

DISCUSSION The results presented on the hydrolysis of linear duplex

DNA suggest the following mode of action of the pure exo-

499

nuclease V of Bacillus subtilis of our preparations in the optimal conditions. The enzyme attaches itself at one end of the DNA substrate and proceed to cleave mononucleotides (or pairs of mononucleotides) one by one from both strands until it degrades the whole segment or it meets another enzyme molecule acting in the same way from the other end. The processive mechanism seems to be a common feature of all bacterial ATP-dependent DNases in onditions similar to those of our experiments [I, 13,18-201. The difference in the B. subtilis enzyme lies in the fact that the limit products are exclusively mononucleotides while in all the other cases they are short-chain oligonucleotides of varying length. On this point however quantitative differences exist as regards the individual DNases. The enzymes of the following organisms yield low but significant proportions of mononucleotides: Mycobacterium smegmatis (4- 13 %) [18], B. laterosporus (6.5 %) [21], Pseudomonas aeruginosa (5 %) [22], M . luteus (1.9%) [21]. The Escherichia coli and H . influenzae enzymes, on the other hand, liberate almost no mononucleotides [13,20]. The B. subtilis DNase therefore seems to be an extreme case.

During the processive exonucleolytic reaction of the B. subtilis DNase described above, ATP is also hydrolysed (3 molecules per phosphodiester bond cleaved). It is likely that the energy released by the hydrolysis of ATP is not utilized in the DNase reaction. From the stoichiometry studies on many ATP-dependent DNases it is apparent that the ATPase and the DNase reactions are not directly coupled. ATP can also be hydrolysed by certain of these enzymes in conditions where there is no DNA degradation [16,28]. The requirement for ATP when this class of enzymes act only as exonucleases on duplex DNA is therefore not clear. It is possible that attachment of ATP is necessary to create the active conformation of the enzyme molecule even for the DNase reaction and that this attachment results in ATP hydrolysis. Under conditions different from those of our experiments (high ATP concentration, less alkaline pH etc.) many ATP-dependent DNases display unwinding activity on double-stranded DNA and the energy released by ATP hydrolysis should be used in this reaction [29,30]. Shemyakin et al. [I51 have recently reported results suggesting that unwinding and cleavage of long single- stranded segments may occur with their preparations of the B. suhtilis enzyme. Eichler and Lehman [31] have postulated that the formation of single-stranded regions in a DNA molecule may reflect the physiological function of the E. coli ATP- dependent DNase in the recombination process. On the other hand studies on temperature-sensitive recombination-defective mutants of E. coli which lack only the exonuclease activity on double-stranded DNA at the non-permissive temperature suggest that this activity is the most significant in vivo

The involvement of the ATP-dependent DNase in genetic recombination is, however, not yet established in all organisms studied. In E. coli the recB and recC mutants (lacking this activity) are generally considered as recombination-defective [35,36]. It is less clear for H. influenzae and B. subtilis. Similar mutants of H . influenzae are little affected in transformation [37,38]. Results obtained in this laboratory (Sedgwick and Anagnostopoulos, unpublished data) indicate that in B. subtilis mutants devoid of ATP-dependent DNase activity the effect of the mutations on transformation and transduction

[32- 341.

frequencies can be, at least partially, explained by their effect on cell viability.

This work was supported by contract 225-BIO F of the Commission of the European Communities.

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J. Doly, D. Le Roscouet and C. Anagnostopoulos, Centre de Gtnetique Molkculaire du Centre National de la Recherche Scientifique, F-91190 Gif-sur-Yvette, France

Documents

Substrate Specificity and Adenosine Triphosphatase Activity of the ATP-Dependent Deoxyribonuclease, of Bacillus subtilis