Characterization of a deoxyribonuclease of Mustelus canis liver

BIOCHIMICA ET BIOPHYSICA ACTA

CHARACTERIZATION OF A DEOXYRIBONUCLEASE OF MUSTELUS CANIS LIVER*

HILARY ASHE, EDNA SEAMAN. HELEN VAN VUNAKIS AND LAWHENCE LEVINE Grad uate Departm ent of B iochemistry, Brandeis Un iversit y , Walth am , and the M arine B iologica Laboratory, 'Woods Hole , Mass. (U .S.A .J (Received October aoth , 1964)

SUMMARY

The presence of an enzyme which hydrolyzes thermally-denatured T4 DNA has been demonstrated in extracts obtained from Mustelus canis livers . The enzyme was detected by its ability to decrease the serologic activity of thermally-denatured T4 DNA when tested with experimentally produced antibodies to denatured T4 DNA. This unique assay was used during purification and characterization of the enzyme. Thermally-denatured DNA was shown to be the preferred substrate for th e enzyme. Selective destruction of denatured DNA by the enzyme was demonstrated when mixtures of native and thermally-denatured T4 DNA were exposed to the nuclease and then subjected to CsCIl! centrifugation. The activity towards thermally-denatured DNA was optimal in o.r M NaCl. It showed a requirement for divalent metals which could be satisfied by Mg2.+, Mn 2 +, and COHo The nuclease has a pH optimum of 8.0. The rate of hydrolysis of thermallydenatured DNA suggested that the digestion proceeded endonucleolytically .

INTRODUCTION

Thermally-denatured DNA is the preferred substrate of a number ofnucleases : the E. coli phosphodiesterase-, micrococcal nuclease-, lamb-brain phosphodiesterase", and Neurospora deoxyribonuclease II. The E. coli phosphodiesterase and Neurospora deoxyribonuclease II are exonucleases while the micrococcal nuclease and the lambbrain phosphodiesterase are endonucleases. The availability of experimental antibodies to thermally-denatured T4 DNA (ref. 3) made possible a survey of various tissues for enzymes capable of hydrolyzing denatured DNA. In a preliminary report, LEVINE AND VAN VU NAKIS Gfound that extracts of Mustelus canis liver had IO-IS times more activity towards thermally-denatured • Publication No. 351 from the graduate Department of Biochemistry, Brandeis University. B ioch im, Biophys. Acta, 99 (I965) 298-306

DNAas e

OF

M ustelus canis

LIVER

DNA than native DNA. Therefore, this tissue was chosen for further purification of the enzyme. The unique assay introduced by HEALY et al. s was used for the characterization of the enzymic activity. This assay measur es the loss in fixation of comp lement (C/) by the immune syste m, as the ant igen (thermally-denatured T4 DNA) is hyd rolyzed by the nuclease into fragments which are t oo small to fix C . MATERIALS AND METHODS M aterials Nuclei c acid s. T4 bacteriophage DNA was prepared from osmotically-sh ocked phage which was subj ect ed to three detergent tre atments and three phenol extracti ons followed by dialysis against isotonic saline". DNA was thermally denatured at 10 /Jog! ml by incubation at 100° in a stoppered tube for 5 min followed by quick chilling. Antibodies to DNA. Experimental antibodies to denatured T-even bacteriophage were used for the immunological assay2.5. The antibody was used at a 1/40 000 dilution and peak C' fixati on occurred at 0.02 f.lg DNA. M ustelus canis livers. These livers were collected at the Marine Biological Laboratory. Woods H ole. Mass. Th e livers were st ored frozen until used. M ethods S ephadex G-200 . Sephadex G-200 (P harrn acia, Lot No. TO 3314) was dispersed in distilled wate r an d the "fines" were decanted afte r t he gel ha d settl ed. Quantitative complemen t (C') fixation. This was car ried out b y the method described by WASSERMAN AND LEVINE? The serological buffer has b een described by TASHJIAN et' at.s and is composed of 0.01 M Tris..:.BCI (pH 7.4). 0.14 M Na .Cl, :> ' 10-4 M MgS0 4 • I,5 ' 10- 4 M CaCI2 • and 0.1 % bovine serum albumin. Immunological procedure for estimating deoxyribonuclease activity. The deoxyribonuclease activity was measured in a manner similar to th at described by HEALY et al. s. The reacti on mixture contained 0.1 ml of an appropriate dilution of enzym e ; 1.0 p g of eit her thermally-denatured or native T4 DNA in a final volume of L a ml cont ai ning 0.01 M Tris-HCI buffer (pH 7-4 or 8.0). O.I4 M NaCl and 1.5 . 10-3 M MgS0 4 or MnCl2 An aliquot of th e reaction mixture was with drawn after I h at 37° and diluted ro-fold in ice-cold C' fixation buffer to terminat e the reaction and to lower the DNA concentration t o a level suitable for the immunological assay. In order to observe serological activity when native DNA was the sub strate, the sample was diluted in C' fixation buffer wh ich did not contain added protein and then thermall y denatured. One unit of enzyme h as been defined as the qu antity of enzyme required t o cause a 50 % loss of ma ximal C' fixat ion after I h at 37°. 0

P urification of the enzyme Frozen M ustelus canis livers were th awed overn ight in the cold room. All operations, except where st at ed, were performed at 4°. Th elivers (about a 10-1 volum e) were homogenized in a Waring Blendor with an equal volume of buffer comp osed of 0.01 M Tris-Hf'l (pH 7.4), 0.14 M NaCl, and 1.5' ro- S M MgS0 4 • The hom ogeniz ed tissue was allowed to stand overnight. The hom ogenate was centrifuged at 8000 X g for 20 min. The supernatant Bi ochim , B iopby s. Act a. 99 (I g65) 298- 3 06

3°0

H. ASHE

et al,

layer, which was below a lipid layer, was removed with a needle and syringe. The 8000 X g supernatant was then centrifuged at 30 000 X g for 30 min. The supernatant fraction was removed with a needle and syringe and stored frozen. Approx. 4 1 of 30 000 X g supernatant were obtained. The 30 000 X g supernatant was thawed and dialyzed against 25 1 of distilled water for 24 h with four changes of dialysis fluid. Any insoluble material which formed was removed by centrifugation at 8000 X g for 15 min. The resulting supernatant was heated for I hat 450 and the precipitate which formed was removed by centrifugation at 8000 X g for 30 min. The supernatant (41) was lyophilized and yielded 45 g of powder which was stored in the deep freeze. Chromatography on Sephadex G-200 was performed at 22° on a 90 X 8 em column which had been equilibrated with distilled water. 10 g of lyophilized powder were suspended in 130 ml distilled water and centrifuged at 28 000 X g for 2.5 h (Spinco Model L, No. 30 rotor). The clear supernatant fluid (120 ml) was removed from the centrifuge tubes and carefully layered onto the top of the column and allowed to sink into the Sephadex. After the sample had entered the Sephadex, a column of water was carefully layered above the Sephadex, The sample was eluted from the column with distilled water. The enzyme which hydrolyzes thermallydenatured DNA was not associated with a distinct protein peak, as measured by the absorbancy of the effluent fractions at 280 mp. Activity towards thermally-denatured DNA was not observed until 2030 ml passed through the column and then was eluted until 3230 ml of water passed through the column. The fractions containing activity towards thermally-denatured T4 DNA (2030 ml to 3230 mI) were pooled and lyophilized. The powder was taken up in 50 ml water and stored frozen. This solution was used to characterize the nuclease activity. Protein concentrations. These concentrations were estimated by determining the absorbancies at 280 and 260 mll and calculating the protein concentrations from the table prepared by LAYNEo. Density gradient centrifugation in CsCI2 . This was carried out by the method of MESELSON, STAHL AND VINOGRAD 1 0 . The DNA was centrifuged at 44770 rev.jmin for 20 h and the banded DNA was photographed using ultraviolet optics. A sample of DNA of known buoyant density (fully deuterated Pseudomonas aeruginosa, buoyant density, 1.763 gjml) was added as a density marker. All buoyant densities in CsClz are referred to that of E. coli DNA which is taken as 1.7IO gjml. The band pattern was traced using a Joyce-Loebl Microdensitometer,

RESULTS AND DISCUSSION

The data in Table I show the enrichment of the preparation with respect to enzymic activity towards thermally-denatured DNA. The heating step was employed because preliminary experiments indicated that the activity toward native DNA is more labile to heat than the activity toward denatured DNA. Purification of the enzyme after chromatography on Sephadex is being attempted but large losses of enzymic activity have been observed. The effect of NaCl concentration on activity toward native and thermallydenatured T4 DNA was tested (Fig. r). The activity toward native DNA is optimal Biochim: Biophvs, Acta, 99 (Ig6S) 298-306

DNAase

OF

Mustelus canis

LIVER

301

TABLE I PURIFICATION OF A NUCLEASE FROM DENATURED D N A

Mustelus canis

LIVER WHICH HYDROLYZES THERMALLY-

Reaction mixtures contain 0.1 m l of an appropriate dilution of enzyme pl us 1.0 pg T4 DNA (n at iv e or denatured) in 0.01 M Tris-Ht.l (pH 7' 4), 0.15 M NaC I, and 1.5' 10- 3 M MgS0 4 ill a final v olu me of La ml. Enrichment is expressed as ratio of acti vity vs. thermally-denatured DNA to activity us. native DNA .

Protein

Enzym e fract ion

[mglml}

Total volume (ml)

Acti vity (tmitsJmg) VS.

native DNA 30 000 X g supern a t a n t Dialyzed and h eat ed 30 000 X g supernata nt 28 000 X g supernatan t of 10 g of lyo p hilized powder" Pooled Sephad ex eluate after lyophilization • The 10 g of powder represents

Enrichment

vs. thermally denatured DNA

32

4000

3. I

37-4

12

16

4°°0

1.5

50.0

33

50

120

0.'f4

43,4

97

30

50

266 , 0

lI5

22%

2·3

of the total powder obtained.

Therm ally denatur ed

10 0

/

T4 - DNA

80

60

40

a

a

20

0 001

3

0 .05 0 .07 0 .08 0.11

0:13

0.15

Nael (M)

Fig. r. Effect of NaCI on deoxyribonuclease activity toward native and thermally-denatured DNA. Reaction mixtures contain : o . r m l enzyme solution plus I.Ottg T 4 DNA (native or thermallydenatured) in La ml of 0.0 1 M T ris-HCI (p H 7.4), 1.5' 10-8 M MgSO•. and the indicated amount of NaC\. IOO% activity corresponds to 9000 units per m l for thermally-denatured T4 DNA and to 470 units per ml for native DNA. • --e. thermally-denatured DNA; 0 - 0. native DNA.

B iochim , B iophys. Acta, 99 (1965) 298-306

H. ASHE

30 2

et at.

at low NaCl concentrations, while activity toward thermally-denatured DNA is maximal at NaCl concentrations of o.OBS-O.Il M. It is import ant to note that in Fig . I maximal activity towards native T4 DNA corresponds to 470 units per ml enzyme solution. while maximal activity toward thermally-denatured T4 DNA corr esponds to 9 000 units per ml enzyme solu tion . To minimize activity towar d native DNA the enzyme was assayed at a final NaG concentration of 0.14 M. The heat stability of the enzyme which hydrolyzes thermally-denatured T4 DNA was t ested in the presence an d absence of 0.1% b ovine serum albumin (F ig. 2).

100

80

2' 60

+ 8SA(0.10f0)

'c '0

...~

%'

40

~u ... 3:.

20

:ec

00

10

20

30 40 50 Temperature

60

70

BO

Fig. 2. Stability of nuclease activity toward thermally-denatured T4 DNA to heat in the presence and absence of added protein. The enzyme (r/roo dilution) was held at indicated temperature for 30 min. ESA, bovine serum albumin.

A IIIOO dilution of the enzyme solution was held at the indi cated temperature for 30 min, chilled in ice and diluted for the immunological assay. In the presence of O.I% bovine serum albumin IOO % activity corresponded to I 200 enzyme units per O.I ml , while in the absence of added protein IOO % activity corresponded to 800 enzyme units per O.I ml , The presence of added protein partially protected the enzyme against thermal denaturation. The metal requirements of the enz yme hydrolyzing denatured T4 DNA were tested under the conditions described in Table II. Each met al was tested at three concentrations (ro- 2 M, IO- 9 M and 10-4 M) and only tho se which showed activity at a r /800 dilution of enzyme were titrated to an end-point. The active metals Biochim: Biophys. Acta, 99 (19 65) 29 8-306

DNAase

OF

M usteius canis

LI VE R

3°3

TABLE II METAL REQUIREM E NT t-O R E N ZY MI C ACT IV I T Y T OWA R D THERMAL LY-D E NA T URED

T 4 D NA

T he following m etals s howed little or no enha nce ment of activity w hen tested wit h a 1/800 dilution of enzy me: Ca2+, BaH , Sr H , CdH , Zn H , an d Cu'+ , Me tal

Optim al concentration (M X I Oa)

M g'k Mn H

1.5

7

200

z2 5 00 19 000

1.5 1.0

C OH

Activity (un itslml)

(magnesium, manganese and cobalt) showed optimal activity at 10- 8 M. Man gan ese and cobalt enhanced the enzymic act ivity 3-fold as compared to magnesium. The pH depen dence of the nuclease acti vity toward native or thermallydenatured DNA is pr esented in Fig. 3. There are two maxima for a ctivity toward denatured DNA, one a t pH 4.5- 5.0 and the other at pH 8.0; while the activity t oward the native D NA has a pH optimum at pH 4.5-5 .0. It is clear that one can redu ce the activity t oward native DNA by performing the assay at p H 8.0. To determine if the activi ty observed at pH 5.0 against both native and t hermally-denatured DNA was due t o one enzyme which did not distinguish b etween 1000on..---

- - --

-

-

-

-

-

-

-

-....

100 0

10 00

x

..

>,

.-

'S;

:;; u 0

1lI

E

100

>,

T4 - DNA

N

~

x ............x

.-Ul

'e :;)

\..

10

\

............

x

13.0

4.0

5 .0

.0

7.0

8 .0

ao

10.0

pH F ig . 3. E ffect of pH on nuclease act ivities to ward native an d t hermally-denatured D NA. For pH 3,4, 4.4 and 5 .2 a cetic ac id-sod ium acet ate bu ffer was used ; for pH 5.2-8. 6, Trts-maleate buffer, and for pH 8.6 and 9.2, gly cin e- N aOH buffer.

B i ochi m , B iop liy s. A cta, 99 (1965) 298- 3°6

H. ASHE

et al,

TABLE III COMPARISON OF THE RATIO OF ACTIVITIES TOWARD THERMALLY- DENATURED AND FROM pH 3.6 TO 5.8 WITH CRUOE AND PURIFIED NUCLEASE P RE PA RAT ION S

NATIVE

DNA

Experimental conditions are th e sa m e as in Fig. 3 . The ratio is expressed as units pe r tnt vs . therma lly-denatured DNA/ units per rnl vs. native DNA.

pH

Ratio

30 000 X g supernatant

3. 6 4·4 5.2 5. 8

Pooled Sephade» eluate after lyophilization

0·5

0.4- 8

1.2

0·73 0.63 4. 2

1.1 14

the two forms of DNA, a pH curve was run under identical condi tions as those presented in Fig. 3 with a crueler p reparation of the enzyme (the original 30 000 X g supernatant). The results (Table III) showed different relative amounts of each enzymic activity from pH 3.6 to 5.8 and indicated that the activity seen at pH 5 was

No enzy me

j

Enzyme (1/1000)

v

u

c

o

.0 L

o
.0

«

Enzy me <1/100)

1.763

Dens ity Fig. 4. Banding of native and thermally-denatured T4 DNA in esC!s- The reaction mixtures contained 14.3/-tg native T4 DNA and 14·3 /l-g thermally-denatured T4 DNA in r .o ml of 0.01 M Tris-HC! buffer (pH 8.0)' 0.14 M NaCl, l.5 · 1 0 - 3 M MnC!. and 0.1 ml enzyme of the indicated dilution. They were in cu bat ed for I h at 37°. Aliquots (0.22 m l) of each reaction mixture were added to 0.83 ml 5.7 M esCl s- D enatured Ps. aeruginosa DNA (0.5 ,ug) was added to each sample prior to centrifugation. Biochim , Biophys. Acta, 99 (196 5) 298-306

DNAase

OF

MHstelHs canis LIVER

not due to one enzyme which did not dist inguis h between native and thermallydenatured T4 DNA, but probably was due to two enzymes . The specificity of t he enzyme activity t oward thermally-denatured T4 DNA is shown in Fig. 4. In these experiments artificial mixtures of equal amounts of native and thermally-denatured T4 DNA were incubated with the enzyme for I h at 37° and then subjected to density-gradient centrifugation in CsCl2 . The figure shows that the native DNA band was not changed after exposure of the DNA to a 1/100 dilution of the enzym e while the denatured band has been partially destroyed after exposure to a 1/1000 dilution of enzy me. At the highest concentration of enzyme used, there was no destruction of native DNA. The data in Fig . 5 show t hat the decrease in C' fixation occurs without a pronounced lag at three different concentrations of enzyme. The decrease in C' fixation 10~=
01

c:

'c '0

6

Enz(1/4000)

E III L

0

'5

~

"v

'c III

Enz (1/ 1000)

Ol

:OJ

c

o

2

Enz( 1/2000)

0

0-

°0

10

20 30 Tim e (m in)

40

50

Fig. 5. Rate of cleavage of thermally-denature d T 4 DNA by varying concentrat ions of Mustetus 0.1 ml enzyme of indicated dilution plus s.o ug thermally-denatured 1'4 DNA in I ml of 0.01 M Tris-HC! (pH 8 .0) ,0.14 M NaC! and 1.5 ' 10-· M MnCl s- The amount of C' fixed at zero time represents 100% activity.

c:artis liver enzyme (Enz) . The reaction mixtures contain

was also shown to pro ceed without a lag for lamb-brain phosphodiesterase", an endonuclease whose enzymic products were oligonucleotides varying in chain length from 5 t o 14 residues. In contrast, the decrease in C' fixation after incubation with E . coli phosphodiesterase", an exonuclease, was accompanied by a long lag period": The linear decrease in C' fixatio n of thermally-denatured T4 DNA after t reatment with the nuclease suggests that the enzyme is an endonuclease. Biochim. Biopbys , Acta, 99 (1965) 298-306

H. ASHE

et at.

ACKNOWLEDGEMENTS

This work was supported in part by research grants from the American Cancer Society (C-222) and the National Institutes of Health (AI-oI940-o6). H.A. is a postdoctoral fellow, National Institutes of Health Training Grant Tr GM-212-o5. REFERENCES I 1. R. LEHMAN, I- Biol, Chem., 235 (1960) 1479. z M. L. DIRKSEN AND C. A. DEKKER, Biochem. Biophys, Res. Commun., 7. (1960) 147. 3 ]. W. HEALY, D. STOLLAR, M. 1. SIMON" AND L. LEVINE, Arch. Biochem, Biophys., 103 (1963) 4 6 r. 4 J. LINN AND 1. R. LEHMAN, Federation Proc., 23 (196 4) 373. 5 L. LEVINE, W. T. MURAKAMI, H. VAN VUNAKIS AND L. GROSSMAN, en». Natt. Acad. Sci. U.S., 46 (1960) 1038 . 6 L. LEVINE AND H. VAN VUNAKIS, Bioi. Bull., 125 (1963) 384. 7 E. WASSER.MAN AND L. LEVINE, J. Lmmunol., 87 (1961) 290. 8 A. H. TASHJIAN, L. LEVINE AND P. 1.. MUNSON, j. Exptl, Med., Ilg (1g64) 467. 9 E. LAYNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 447. 10 M. MESELSON, W. W. STAHL AND ]. VINOGRAD, Proe. Nati, A cad. Sci. U.S., 43 (1957) 581. II W. T. MURAKAMI, H. VAN VUNAKIS, L. GROSSMAN AND 1.. LEVINE, Virology, 14 (1961) 190.

Bioehim. Biophys. Acta, 99 (1965) 2g8-306