Calcium protects DNase I from proteinase K: A new method for the removal of contaminating RNase from DNase I

ANA1

YTICAI.

107, 260-264 (l%io)

BlOCHtMlS~lRY

Calcium

Protects DNase I from Proteinase K: A New Method Removal of Contaminating RNase from DNase I H. TULLIS

RICHARD

Received DNase

I and

polymerized while in

the

two

enzymes

of up to 1 mg/ml. RNase by proteinase K in the

proteinase

by rRNA centrifugation. in

K are

March

and

K in the poly(A-j These

presence

proteinase K nating RNase.

presence

of Ca”

to

obtain

of

Ca”’

of Ca”’

better DNase

1980 used

in the purification

DNase I is rapidly inactivated to proteinase K inactivation

selectively

removed

RNA degradation ti) DNase A and digestion can

Cast is required for the activity of DNase I against Mg’+ DNA at pH 7.5 (1). Ca” is also known to stabilize a number of enzymes including DNase I to protease action (2,3). In the course of initiating a routine procedure for isolating highly polymerized RNA, we noticed that Ca” completely stabilized DNase 1 to proteinase K attack. The remarkable stability of Cast DNase to proteinase K suggested (i) that DNase and proteinase K could be used together rather than sequentially in isolating highly polymerized RNA, and (ii) the possibility of removing the traces of RNase A which are often found in commercial preparations of “RNase-free” DNase. The results presented here support these suggestions. EXPERIMENTAL

28.

PROCEDURE

Mr~tcrials. DNase I (Worthington. DPFF grade), RNase (Sigma, DNase free), proteinase K (EM Biochemicals), and Pronase (Calbiochem) were stored at -20°C. ]5”-HIUridine (25 CJmmol) was purchased from New England Nuclear. [“H]Polyuridylic

RNase

monitored proteinase

of nucleoprotein be

used

to

DNase of DNase

A activity

as judged

by sucrose K can be used

gradient together

complexes.

selectively

of highly

by proteinase K even at protease

A. a common contaminant of “RNase-free” presence or absence of Ca”. I’reatment

RNA ribosomal results suggest that

treatment

RLJBIN

HARVEY

commonly

RNA. In the presence of EDTA 10 rnh< Ca” DNase is totally immune

concentrations was inactivated with

proteinase

AND

for the

remove

and

1

(ii)

contami-

acid was obtained from Collaborative Research. Ultrapure Tris and sucrose (RNase free) were from Schwartz/Mann. TAME’ (p-toluene-I.-arginine methyl ester) was purchased from Worthington. All other chemicals used in these experiments were reagent grade. The glassware used in all experiments was baked at 230°C for 4 h to remove all possibleenzyme contamination. RNA Irrhrliwg rrtld prrri~c,rrtiotr. RNA and DNA were purified from sea urchin (Tripnru,stcs ,grtrtilltr ) eggs and hlastula-stage embryos according to Kleene and Humphreys (8). with the modification that DNase digestions were carried out in 2 mF*i CaCl, and IO rnM MgCl?. The inclusion of Ca”- in the digestion buffer is necessary for optimal activity since Ca” is required for DNase I activity at pH 7 (I). Pulse-labeled poly( A +)-mRNA was isolated from early blastula embryos grown in [“Hluridine (I C/ml in filtered seawater) for 1 h at room temperature and subse’ Abbreviations methyl ester):

SDS.

used: ‘I AME t/l-toluene-t \odium dodecyl sulfate.

-argtnyl

Ca”

0

FIG. DNase

15

I.

The

effect

1 to proteinase

PROTECTS

45

30 Tnme (m4nJ

of

calcium K.

DNase

on

the

I (100

rnM Tris. pH &/IO rnkt CaCI, was incubated teinase K ( I mg/ml) at 37°C. After I h EDTA to 15 rnM and the ashy was repeated.

DNase

60

stability &ml

of in 20

with prowas added

quently purified on oligo(dT)cellulose columns as described (10). The specific activity of the RNA was determined using a molar absorptivity constant for RNA-P at 260 nm of &, ,,,,, = 280. Poly (A)-containing RNA was assayed by poly( U) hybridization as described by Duncan (19). En:~~n~ c~ssrt~s. DNase activity was determined using sea urchin sperm DNA by the hyperchromicity assay of Kunitz (5) as modified by Price cut ((1. (20). Pronase and proteinase K activity were determined from the rate of hydrolysis of TAME (4). RNase activity was determined by hyperchromicity (21) using purified 26 S ribosomal RNA (4 pgiml in 5 mM sodium phosphate buffer, pH 6.8). Itwcti~~crtim t?f‘ DNax L~II~ RNase lrsitlg protritusc K. DNase was tested for stability to proteinase K in the following manner. In a typical experiment, DNase was dissolved to 100 pgiml in 20 mM Tris buffer (pH 7.5) containing 10 mM CaCI, and equilibrated at 37°C for 20 min. At the end of the test period, the solution was made to 1 mg/ml in proteinase K and the incubation continued. Aliquots were taken at appropriate intervals to test DNase activity. The solutions were then made 15 mM in EDTA using 0.5 M EDTA (pH 7.5) and the incubation continued with aliquots taken as neces-

FROM

361

PROTEIN.ASE

sary to follow the course of inactivation. RNase digestions with proteinase K were carried out in the same manner. Sttrhility of‘ proteintrsr K cltld Plor~c~~c. The stability of proteinase K and Pronase was measured at 37°C and 1 mg protein/ml in 30 mM Tris, pH 8. containing 20 mM CaCl,. R~~tno\~rrl c!f’ RNtrsc crcti\‘itx ,jht~l DNlr.vc, 1 usitlg protvitlrrxc~ K. DNase was made to 1 mg/ml in 20 mM Tris buffer, pH 7.5. containing 10 mht CaCl, and equilibrated at 37°C. The solution was then made to I mg/ml in proteinase K and incubated for I h at 37°C. The solution was then tested for RNase activity by incubating an aliquot with either poly(A- )-mRNA or total blastula RNA for I to 3 h at 37°C. At the end of the test incubation. the solution was made to I5 rnbr EDTA and 15%SDS and the incubation continued for 1 h to remove all remaining traces of nuclease activity. Phenolchloroform extraction was then carried out to remove proteinase K and the resultant solution centrifuged on SDS-sucrose (520%) velocity gradients to determine the degree of degradation (6.X). RESULTS TAP

c,fjbcl

c7.f’ Co’*

of1

111~~ sfrthilifx

c!f’

DNrrsr I protrirlrrsc K. Figure I shows the effect of Ca” on the stability of DNase 1 in the presence of 1 mg/ml proteinase K. In 10 rnM Ca”+ DNase I activity is stable to proteinase K. The removal of Ca”. by the addition of 15 mbt EDTA caused DNAse activity to decay rapidly. A logarithmic plot of the rate of inactivation was linear with time up to 90% inactivation (data not shown). The half-inactivation time under these conditions was 10 min. SDS was not used in these digestions since SDS at low concentrations inhibitors DNase activity. as well as forms insoluble calcium salts ( 1 I). The observed stabilization of DNase 1 to proteinase K by Ca” could be due to a Ca”-induced inactivation of the protease. That this is not true is shown in Fig. 2. Pro-

262

TULLIS

AND

teinase K was both stable and highly active in 10 mM Ca?+. Under these conditions the half-inactivation time was about 60 h. In contrast, the protease activity in Pronase shown in the same figure had a half-life of less than 8 h. The activity of proteinase K was the same in the presence and absence of Ca’+. Thr c)Jfixt of Ctr” OH tlw stability of RNtrsr A to proteintlse K digestion. Brisson and Chambon (6) and others have shown that commerical preparations of RNase-free DNase are contaminated with substantial amounts of RNase activity (-0.0 1% RNase A equivalents by weight). In order to determine whether or not proteinase K could selectively inactivate RNase A in the presence of Ca?+ we measured the stability of RNase to proteinase K in the presence of Ca”‘. The results shown in Fig. 3 demonstrated that 50 pg/ml proteinase K rapidly inactivated RNase A at 37°C in the presence of Ca’+. A log plot of the rate of inactivation was linear with time up to 98% RNase inactivation. The half-reaction time was 20 min under these conditions. Rt~mo~‘rrl ofRNtIst> rrctivity .fkom RNtlw ,fktJ DNtrsr by proteirltrsr~ K digt>stiotl in thr prcsencr c!f’C’rr’+. To test the effectiveness of proteinase K in removing RNase activity from RNase-free DNase. a solution

RUBIN

0

40 Time

FIG. 1. Kinetics K. RNase A (100 m&t CaCI, absence

was (a)

(min)

of RNase inactivation &ml) in 5 mht ‘l’ri\

incubated

of proteinase

1

12 0

80

at 37°C

in the

by proteinaw buffer. pH WI0 presence

(0)

01

K at 50 &ml.

of DNase I in IO mM Cast was treated with 1 mg/ml proteinase K for 1 h as described under Experimental Procedure. This solution containing DNase, proteinase K, and Ca” was tested for RNase activity by incubation with either total blastula RNA (Fig. 4) or purified poly(A+)-mRNA (Fig. 5). In both cases, control solutions not treated with proteinase K caused nearly complete hydrolysis of the RNA to small oligonucleotide fragments. DNase treated with proteinase K in the presence of Ca“+ did not significantly degrade either blastula rRNA or poly(A’)-mRNA. DISCUSSION

FIG. 1. Stability of proteina\e K in the presence of calcium. Protrinase K (0 ~ 0) or Pronase tb - - - A) at I mgiml in 20 mhi Tris. pH X/IO m\t CCII

at 37°C.

Proteinase K is a broad-specificity serine protease isolated from the mold Tritirtrclziutn rrlbunl (7,12- 14), which has found wide application in the isolation of purified nucleic acids (I18). Our results demonstrate that DNase I. which is often used in such isolations. is remarkably stabilized to inactivation by proteinase K in the presence of Ca?+. This cannot be due to an effect of Ca?+ on the stability of proteinase K since the enzyme is both stable and highly active in the presence of calcium. This is consistent with the observation that proteinase

Ca-

PROTECTS

DNa\e

FROM

363

PROTEINASE

make this less likely since proteinase K has a very broad substrate specificity, analogous to that of subtilisin (7.13). Our results also show that DNase I and RNase A are differentially susceptible to proteinase K in the presence of Ca”. Many if not all commerical preparations of RNasefree DNase contain small amounts of contaminating RNase activity (6) which can greatly affect the isolation of intact RNA. Although RNase activity can be removed by other techniques such as chromatography on UMP-Sepharose (6) or Sephadex G-50 (33). these procedures are somewhat time consuming and expensive. The alternative presented here is that RNase activity be removed from DNase by predigestion with proteinase K in the presence of Ca”. The conditions which we currently employ for this are DNase and proteinase K (I mgiml each) in 20 rnbt Tris, pH 8. and IO mhi CaCl, kept at 37°C for 3 h. This solution is then added directly to the homogenate or nucleic acid preparation

FIG. 4. Sea urchin with

proteinase

blastula

total

RNA

RNA was treated with as described under centrifuged on a T-203

proteinaae-K-incubated Experimental SDS-sucrose

(SW 41 rotor, 8 h at 40,000 assayed for total RNA (A?,,,,

rpm. ,1,,, (---I

mRNA

poly-[

by hybridization

after

K-incubated-DNase.

with

treatment

Bla\tula

total

Procedures density

DNase and gradient

25’C). and ‘HI-U

Fraction\ poly(A-)(- - -1.

K is stable to autodigestion in the presence of EDTA (6). Calcium DNase is also stable to chymotrypsin and trypsin (3). The calcium protective effect is due to a Ca’+-induced conformation change which occurs when Ca”+ binds to one or another or both of the two tight Ca”’ binding sites on DNase ( I-3.19). Since chymotrypsin and trypsin have relatively narrow amino acyl specificities. the Ca’- effect could be due to steric constraints imposed by Ca”- binding at or near the sites of protease attack. Our results

:‘:

0 5 Fraction

FIL.

5.

Density

gradient

10 No

15

centrifugation

of

purified

poly(A’ DNase treated

I-mRNA treated with proteinase K-incubated I. Purified poly(A’)-mRNA(untreated 0 ~ with RNase-free DNase (0 - -U) at

@g/ml DNase Sample\

for tl

CHCI,, bottom

I h: ~ were

treated A) at incubated.

and run on of the gradient

with 100

proteinahe /*g/ml for extracted

SDS-sucrose i\ on the

a\ left.

0): 100

K-incubated I h at 37°C. with phenol: in

Fig.

1.

fhe

264

TULLIS

AND

from which purified RNA is to be extracted. After an appropriate incubation with both enzymes, addition of sufficient EDTA to chelate all available Ca”+ and 1% SDS results in the rapid and complete inactivation of the DNase. One major advantage of this procedure is that DNase and proteinase K digestions are done together. This results in a more rapid and complete digestion of DNA-nucleoprotein complexes and reduces the exposure of the RNA to endogenous RNases which occurs during the normal DNase digestion. ACKNOWLEDGMENTS We

would

of Dr.

Tom

this and

lihe

to acknowledge

Humphreys

from and

in whose

work was performed. Roger Duncan for

hybridization assay,. part by Postdoctoral

Research

help

laboratory

and

some

This work was supported Fellowship 5 F 32 HDO5248-0’ and Human grant from

oi

Price.

Poulos. 247, 3. Tullis.

Development) the California

./. B/o/.

C‘lrc,,?/.

250.

917.

T. L.. and Price. P. A. ( lY71)1. Bit)/. C‘hc,rrl. 2900. R. H. (IY75) Ph. D. thesis. University ot San

Diego.

Hummel. B. C. W. ( 1959) Ctrtltrd. Ph~viol. 37. 1393. Kunitz. M. t 19.50) .I. G’erl. Pl~x.xi,d

6. Brisson. 0.. ~~/1<‘/~1. 75, 7.

Ebeling. 91.

and 402.

W..

Chambon.

(I/ (I/.

( 1974)

J.

Bi~~c~/u,,,~.

33.

349.

P. t 1976)

Ano/

brrr.

BICJ-

J. Birwhcm.

47.

x.

Kleene. K. C.. and Humphreys. T. tlY77) Cc,// 12, 143. 9. Tullis. R. H.. and Price, P. .A ( lY74)./. Bicd. C’/fctu. 249.

2523.

IO.

Bantle. J. A.. ( 1976) Am/.

I I. I’.

Liao. Krauh.

13.

Sc~kr‘\ Morihara.

14.

c’llCJ/,l. 39. 14x9. Krau<. E.. Kiltr. M. H., and

1.

H,,ppc~ Gross-Bellard.

Ih.

( 1973) E/rr-. J. Ri,~~hc,/,r. 36. 31. Hilz, H.. Wiegers. U., and Adamietz,

Maxwell. Biochrm.

Wiegerh. 77.

t-1.. and

IX. 19.

Wieger\. /‘/I\\. Duncan.

20

Hawaii. Price. P.

Honolulu. A.. Liu.

Moore.

S. ( lY6Y)

ti.. KC\. R.

Tavernier. c.lroriit/r>

27

Smith. and

W.

E.

357, 937. H. (1975) A,qr. Femfert.

Bitd.

U. F. t 1976)

.S~~~/c~‘, Z. Ph~,s;,,/. C’lrc,,,~. 357, 233. M., Oudet, P.. and Chambon.

17.

11

Hahn,

Clrrm. 250, 383 1. U. F. t 1976) 110[>1)(,

L. P/?Cd. C‘lrrrtr. K.. and Tsuzuhi.

.I. R/fu.llcJtH.

lO3-

I. M., and 70, 4 13.

T. H. ( 1975) ./. Bit)/. F... and Femfert.

Eur.

Foundation.

P. A. t 1975)

California.

5

in

REFERENCES I. 7.

4.

advice

I also thank Mark Boardman their help with the poly(U)

the NIH (Child Health by a special research

Biomedical

the

RUBIN

56, Hilz.

(1971)

Ff
and HilL. H. t 197 I ) Rirxl~c,rrr. C‘~~~rl,rlu/f. 44, 5 I?. (lY7X) Ph.D. thesis. University

M. J.. Hough, Davidson. E.

Y..

J. Bi,d.

P. E.. and 9. 2X46.

IX.

P. 11975)

1t1.3. H.

‘T.

P.

Gray.

Stein, Chrm. E.

W. 244. 0.

23. Bioof

H..

and

917. (1970)

B. R.. Chamherlin. H. t lY74) J. ,ZIo/.

M. Bid.

RioE., 85,