Elimination of residual RNase activity from purified preparations of RNA-directed DNA polymerase

Vol. 67, No. 3, 1975

BIOCHEMICAL

ELIMINATION FROM

AND BIOPHYSICAL

OF RESIDUAL

PURIFIED

RNase

PREPARATIONS

DIRECTED

DNA

RESEARCH COMMUNICATIONS

ACTIVITY OF RNA-

POLYMERASE

Marc

S. Collett and Anthony J. Faras Department of Microbiology University of Minnesota Medical School Minneapolis, Minnesota 55455 Received

September

30,197s SUMMARY

Preparations of RNA-directed DNA polymerase purified from RNA tumor viruses by standard methods generally contain trace amounts of single-stranded RNA endonucleolytic activity detectable only by relatively sensitive methods. This contaminating RNase activity has been found to be completely inhibited when RNA-directed DNA poIymerase reactions are carried out in the presence of low concentrations of bentonite. Under these conditions, only minimal inhibition of the DNA polymerase and RNase H activities of the RNA-directed DNA polymerase was observed. INTRODUCTION The

RNA-directed

has been

purified

of various gel

by a wide

combinations

filtration,

purity

DNA

DNA

presence

of nuclease

interested genome

transcription.

Copyright All rights

DNA

analysis

activities

(other

to elucidate

of RNA-directed

Q I9 75 b.v Academic Press. inc. of reproduction in anv form reserved.

DNA

of protein

RNase

for

polymerase. 946

our

have

consisted

(i-7).

for

We have

been

70s

RNA

oncornavirus mechanisms

However,

by

by sodium

and assays

studies

The

assessed

bands

H) (8,9).

the reaction

viruses

chromatography,

has been

of the avian

prerequisite

of which

electrophoresis,

than

transcription

in an effort

A necessary

gel

tumor

chromatography

preparations

activity,

RNA

most

and affiniQ

polyacrylamide

in the --in vitro into

preparations

(SDS)

with

ion-exchange

fractionation,

polymerase

of specific

sulfate

associated

of procedures,

centrifigation

of oncornavirus

dodecyl

variety

of salt

velocity

determinations

polymerase

is highly relatively

of

the

reverse

purified few

BIOCHEMICAL

Vol. 67, No. 3, 1975

reports

of

of

the

(1

- 4).

extensive

DNA

DNA

polymerase

polymerase

remain

(9).

This

it in

the

a single

evidence

DNA

the

reaction on be

either

detected

g. of in

In

residual

This

the

RNA-directed the

including

presence

MATERIALS

may this

of

appropriate

AND

not

be

most

detectable

alter we

activity concentrations

by when

--in

vitro the

in

that

nature

present

contaminating DNA

concentrations selective

polymerases

critical

routinely

RNA-directed

the

activity DNA

completely

polymerase

from

polymerase

activity

appears DNA

be

purity

endonucleolytic

communication

low

inhibition

may

DNA

RNase

remove

a ribonuclease

may

of avian by

to

which

template

the

addressed

oncornavirus

RNA-directed

preparations

mixtures.

such

purified

reaction. the

difficult

of

which

rigorously

RNA

activib,

RNA

in

been

solubilization),

the

eliminated

in

most

highly

RNase

the

has

amounts

RNA

polymerase

completely

trace

of

(e.

purified

be

can

amount

that

proved

RESEARCH COMMUNICATIONS

appeared

a single-stranded

of even

scission

conventionally

effect

fractions

indicating

can

that

capabilities

chain true

has is

appears

methods

evaluating

the

which

residual

conventional

have obtained

preparations

since

may

purification

preparation

A contaminant

activity

of

enzyme

AND BIOPHYSICAL

polymerases of

since

bentonite little,

or RNase of

the

in if

any,

H activity inhibitor.

METHODS

Reagents. C3H]-thymidine triphosphate (53 Ci/mM) a;d [3H1-poly (rA) (19 mC/mM) were obtained from Schwarz Bioresearch. [ HI-uridine (20 Ci/mM) was from Nuclear Dynamics. The sources and preparation of other pertinent materials have been described previously (1, IO). Cells and virus. The 677 strain (subgroup C) of Rous sarcoma virus (RSV) was propagated in duck embryo fibroblasts and purified as previously described (1 1). The preparation of RSV 70s RNA and [3H]-uridine labeled 705 RNA have been reported previously (11 - 13). DNA polymerase reaction. Avian myeloblastosis virus (AMV) DNA polymerase, obtained from J. W. Beard of Life Sciences, Inc., St. Petersberg, Florida, through the auspices of the Special Virus Program of the National Cancer Institute was purified as described by Kacian and Spiegelman (14). Two preparations were employed in these studies, V74-17, and V75-2. AMV 70s RNA was purified by previously described methods (1 1 - 13). Standard DNA polymerase reaction mixtures contained 0. 1 M Tris:HCl, pH 8. 1; 0.01 M MgC12; 2% (v/v) 2-mercaptoethanol; 6 x 10 -5 P/I unlabeled deoxynucleoside triphosphates (dATP, dCTP and dGTP); 5.9 x 10v5 M unlabeled TTP; 1 x lo-6M

947

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[3H]-TTP; 5 m/ml 70s RNA; 50 units/ml AMV DNA polymerase (I, 15). Reactions (50 ~1) were carried out at 37” for 1 hr and the incorporation of radiolabel into acid-precipitable material was determined as previously described (16). RNase H assay. The RNase H activib was assayed using [3H]-(rA)n* (dT)l216 as substrate. The components of the standard RNase H reaction mixtures-using the AMV DNA polymerase were the same as those for the DNA polymerase reaction described above except that 70s RNA and all dNTPs were omitted and [3H]-(rA)* (dT)l2-18 included. Reactions (50 ~1) were carried out at 37’ for 1 hr and the RNase H activity determined as reported previously (17, Collett and Faras, submitted for publication). Polyacrylamide gel electrophoresis. viral RNA in 2.25% polyacrylamide published (1). The exact conditions legend to Figure 4. Preparation of bentonite. Bentonite previously reported (18).

The gels of

of the viously in the as

was

procedures used were essentially electrophoresis

washed

and

for as are

sorted

the analyses those predescribed

essentially

RESULTS Effect

of

bentonite

on

01

the

DNA

I

polymerase

I 40

BENTONITE

1

and

I 80

CONCENTRATION

1

RNase

I 120

H activities

RNA-

I

lvglml)

Figure 1. Effect of bentonite on the DNA polymerase and in purified preparations of RNA-directed DNA polymerase. merase and RNase H activities were assayed as described and Methods. Each duplicate 50 yl reaction was incubated concentrations of bentonite for 1 hr at 37”. Symbols: 0, DNA polymerase activity 4 RNase H activity

948

of

RNase The

H activities DNA poly-

in the Materials with the indicated

Vol.

67,

No.

3,

directed

DNA

isolation

of

tively

1975

RNAs

of

the

to

effect in

purified

viral

DNA

--in

vivo

DNA

polymerase As

21

can

in

Figure

of

capable

that

the

particular

centration

by

C3H]-labeled of

AMV

complete

DNA

bentonite

70s

subunits is

degradation

the

incubation

949

oncornavirus-

synthesis

of proviral

polypeptide

a gradual

RNase

bentonite

as

the

on

this

and

H activities

template.

with

To

in activity

was

an

in the

Figure absence upon

mixtures,

pre-

monitored containing

of

such

determine

purified

mixtures

(1).

However,

and

to be

RNA

presented

used. the

the

denatured

in

use

polymerase

of

reaction

from

occurred

in

DNA

contamination

was

I

first

concentrations

RNA

polymerase

present

viral

in

DNase

possible

effect

RNase

RNA

of

mixture.

centrifugation

RNA

preparation

the

selec-

investigated

same

reaction

the

varying

rate-zonal viral

RNA enzyme

the

the

and

RNase

and

1 hr,

analyzed

of

eliminating

70s

of

on

the

to

polymerase

in

appears

of

viral

period

preparation

integrity

polymerase

a role

tested

in

also

of

AMV

purified

polymerase

[‘HI-labeled

subunits

seen

of

DNA

bentonite

the

the

the

preparations

since

play

there

potymerase,

DNA

of

on

1,

the of

the

also

DNA

incubation

profiles

both

in

on

reside

RNA-directed

incubating

RNA

of

of bentonite

RNA-directed an

seen

to we

the

therefore

bentonite

to

and

investigate

Furthermore,

appears

be

concentrations

was

and

24),

increasing

bentonite

We

thought

20)

during

preparations

contamination

template. is

used

(19,

to

containing

(3,

inhibition

by

as

- 23)

interest

of

COMMUNICATIONS

occasionally

commercial

RNase

activity

coordinate

Effect

of

RESEARCH

activities

polymerase.

H activity

(8,

was

reactions RNA

RNase

DNA

in

concentrations

70s

been

RNase

residual

reconstructed

associated

parations

it

eliminate

of various

activity.

general

Thus,

BIOPHYSICAL

has

RNases

RNA-directed

activity

if

inhibit

(18).

bentonite

purified

to

contaminating

oc-amylase

AND

Bentonite

polymerase.

remove

and

BIOCHEMICAL

bentonite.

After

and

the

resulting

The

sedimentation

analysis 2. of

with It

can

bentonite

increasing RNA

be with

the subunit

one

con-

Vol. 67, No. 3, 1975

integriw tion

can by

these

the

identical

may

RNase be

the

activities

of AMV

with

DNA

pol

exposure used

RNA

ymerase. to

enzyme

we

and

two to

different

IO FRACTION

and

is

45%

of

the

the

at

control

80

degrada

pg/ml.

values

preparations

to

compared

purity,

of

Under DNA

of

depicted

20 NUMBER

0

950

effects

using

particular,

of

bentonite

viral is

in

in

different

C3H]-labeled

preparations

purified

and

the

integrity

integrity

results

included

against

remained.

respect

have

enzyme

protected

enzyme

template The

obtain

bentonite

different

vary

RESEARCH COMMUNICATIONS

completely

respectively,

that

contamination,

enzymatic

nearly

66%

case may

AND BIOPHYSICAL

when

H activity,

procedures

ribonuclease

is

approximately and

it

and

preparation

conditions,

As

The

maintained

enzyme

polymerase

after

be

BIOCHEMICAL

panel

compared B o/75-2)

by

on

preparations RNA

subunits

in

Figure

showed

3. no

Vol.

67, No.

3, 1975

BIOCHEMICAL

acid-solubilization period

(Table

35). of

of I),

A second

was

included

subunits

RNA weight

in

both

caused

(Table RNA

w/ml

cases

I),

in was

BIOPHYSICAL

28s

ribosomal

appreciable

preparation

viral

at 32

HI-labeled

still

enzyme

radiolabeled

molecular

but

3

[

AND

caused

subunits enzymatic dramatically

RNA RNA

(V74-17)

which

the

RESEARCH

subunit

reactions, improved

a

1 hr

of

However, the

the

when

integriw

(Figure

(Figure solubilization

degradation

3C).

incubation

degradation

demonstrated

complete

(Figure

after

COMMUNICATIONS

of 36,

C).

high

bentonite the

RNA

Under

Figure 2. Integrity of the viral RNA subunits following incubation in the presence of a purified preparation of AMV DNA polymerase. Incubation mixtures (50 l~.l), ,carried out in 0.02 M Tris:HCl, pH 7.4, 0. OlM EDTA, contained 5 pg/ml unlabeled 70s RNA, 9000 cpm of C3H]-labeled RSV 70s RNA (from virus harvested at 4 hr intervals), 50 units/ml AMV DNA polymerase (preparation V7417), and the indicated concentration of bentonite. After 1 hr at 37’C, reaction mixtures were made 0. 1M EDTA, 500 pg/ml predigested Pronase, and 0.5% SDS. After 20 min at 37’, nucleic acids were extracted with STE (0. 1M NaCl, 0.02M Tris:HCl, pH 7.4, 0.01 M EDTA)-saturated phenol. Samples were then diluted into TE buffer (0.02M Tris:HCl, pH 7.4, 0. OlM EDTA), heated to 100’ for 80 seconds, and rapidly quenched in ice water. The mixtures were then layered onto 15-300/o sucrose gradients and centrifuged in an SW50. 1 rotor at 50,000 rpm for 225 min at 4O. A.

6.

Viral RNA subunit profile after incubation in the absence of AMV DNA polymerase, 0; incubation in the presence of AMV enzyme, 0; incubation in the presence of AMV enzyme and 8 Pg/ml bentonite,A. Viral RNA subunit profile after incubation in the presence of AMV enzyme and 32 pg/ml (0) and 80 pg/ml (0) bentonite.

Figure 3. Integrity of the viral RNA subunits following incubation with different preparations of purified AMV DNA polymerase and either the presence or absence of bentonite. Reaction mixtures (50 ~1) were as described in the Materials and Methods for the DNA olymerase reaction, except that ZP all dNTPs were omitted and 9000 cpm of [ HI-labeled RSV 705 RNA was included per reaction. Two different preparations of AMV DNA polymerase were analysed as described in the legend to Figure 2 in the presence (32 pg/ml) and absence of bentonite. A. Viral RNA subunit profile after incubation in the absence of AMV DNA polymerase at 4’, 0; at 37’, 0. 6. Viral RNA subunit profile after incubation at 37” in the presence of AMV DNA polymerase (preparation V75-2) with (0) and without (0) bentonite. C. Viral RNA subunit profile after incubation at 37’ in the presence of AMV DNA polymerase (preparation V74-17) with (0) and without (0) bentonite.

951

Vol. 67, No. 3, 1975

TABLE

RNA AMV

I.

AMV

BIOCHEMICAL

solubilization and enzymatic DNA polymerase preparations.

DNA polymerase preparation

percent enzymatic DNA

v74-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

V75-2

in

of control activitya

polymerase

17

activities

percent RNase

solubilization of RNAb

H

93%

82%

13%

78%

63%

0%

a

DNA polymerase and RNase H activities were determined as described in the Materials and Methods in the presence of 32 pg/ml bentonite and compared to control activities in reactions lacking bentonite. b3 [: HI-labeled ribosomal 28s RNA was exposed to the enzyme preparations for 1 hr at 37’C. The acid-insoluble material remaining was compared to that in control incubation mixtures lacking enzyme.

these are

conditions only

of incubation,

slightly

In an effort degradation, were

greater

resolution

when

polymerase 32 pg/ml profile

RNA

was

(preparation with

was

V75-2).

the enzyme,

detectable

from

acids

though

employed

incubated

RNase

H activity

of limited

described

gels,

than

sedimentation.

rate-zonal

was

(Table

I),

in the presence However,

when

no solubilization extensive

RNA

3

which As

of RNA degradation

of the purified

AMV

bentonite

included

difference

of the control

RNA

in Figure

polyacrylamide

there

no significant that

and

the detection

as those

in 2.25%

even

periods

for

mixtures

of nucleic

4B,

the incubation

resulted

the sensitivity

incubation

in Figure

DNA polymerase

I).

by electrophoresis

can be seen during

(Table

to increase

similar

analyzed

affords

inhibited

both

was

in the RNA

lacking

DNA

DNA at

electrophoretic

polymerase

(Figure

4A, C). DISCUSSION The

RNA-directed

DNA

polymerase

of oncornaviruses

952

has been

purified

Vol.

67, No.

BIOCHEMICAL

3, 1975

AND

DISTANCE

Figure 4. electrophoresis. in Figure precipitated buffer

of

ribonuclease

of

the

3.

Tris:HCl, in 100

of

pH 7.4, ~1 TE buffer

techniques.

(mm)

DNA

polymerase RNA at

low

appear

concentrations

gel described were in TE

EDTA). heated to 100” for 60 seconds, quenched, gels of 2.25% polyacrylamide, in 6 x 126) mm [ PI-labeled 28s and 18s ribosomal The incubation conditions were as follows: 1 hr at 37’. and AMV DNA and

to

AMV of 32

remain

(nicking) in

DNA kg/ml

when

polymerase

(preparation

V75-2),

polymerase bentonite.

(preparation

V75-2),

rigorously in

In this

(1,4,9). degrading

as

0. OlM were

However,

activity

contaminating bentonite

COMMUNICATIONS

of the viral RNA subunit integrity by polyacrylamide Reactions (50 PI) were carried out for 1 hr at 37’ After Pronase digestion and phenol extraction, mixtures with ethanol, centritiged and the precipitates resuspended

and analyzed by electrophoresis GmA/geI, 4 hr at room temperature. RNAs were included as markers. A. [3H]-Iabeled viral RNA, [3H]-labeIed viral RNA B. 1 hr at 37’. C. C3H]-labeled viral RNA 1 hr at 37’ in the presence

a variety

MIGRATED

RESEARCH

Analysis

(0. 2M Samples

by

BIOPHYSICAL

even

assayed, highly

communication activity

incubation

may mixtures.

purified we

be

trace

preparations

report

eliminated Under

amounts

these

that

this

by

including conditions

Vol. 67, No. 3, 1975

the

DNA

only

BIOCHEMICAL

polymerase

minimally

ducted

in

and inhibited.

the

the

H activities

Thus,

complete

investigating reaction

RNase

absence

out

in

the

the

presence

enzyme

preparation

synthesis

degradation. avian

of

RESEARCH COMMUNICATIONS

DNA

of template of

the

of

RNA-directed

characteristics

carried

AND BIOPHYSICAL

are

may

We

are

RNA-directed

be

con-

currently

DNA

polymerase

bentonite.

ACKNOWLEDGEMENTS

Grant

This No.

investigation CA 14790-01

was supported by from the National

Public Cancer

Health Service Institute.

Research

REFERENCES 1.

Faras,

A. J., Taylor, J. M., J. M. (1972) Biochemistry

2.

Kacian,D. L., Biophys.

3.

Grandgenett, Sci.

D., U. S. A.

Gerard, G. S., 70, 230-234.

4.

Hurwitz,

J.,

Leis,

5.

Marcus,

Watson, Acta 246,

and

S.

L.,

McDonnell, J., 11, 2334-2342.

K. F., 265-383.

J. P.

Modak,

M.

A.,

Burny,

and

and

J.

and

Cavalieri,

W.

Spiegehan,

Green,

(1972)

J.,

Levinson,

M.

Viral.

(1973)

9,

E.,

and

S.

(1971)

Proc.

Bishop,

Biochim.

Nat.

Acad.

Viral.

14,

116-129.

L. F.

(1974)

J.

853-859. 6.

Livingston, Virology

7.

Gerwin, 69,

8.

Green, 14,

9.

Temin, IO,21

IO.

Faras,

D. M. 50,

, Parks, 388-395.

B. I., and 2599-2603. end M., 187-334. H. M., l-235,

Milstein,

and

Faras,

A. J., 859-863.

12.

Collett,

M. 436-445.

S.,

and

G. F.

Mizutani,

A. Viral.

Dibble,

Kieras,

P. , Scolnick,

(1974)

(1974)

12,

Levinson, 334-342.

N. A.

R. M.,

(1975)

and

Proc.

Prog.

S.

C.,

E. M.

(1972)

J. B.

Gerard,

A. J. , Garapin, H. M. (1973) J,

11.

W.

Faras,

954

in

, and

Nat.

Nucl.

The

W.

E.,

Proc.

Acad.

Acid

Enzymes

J.

Acad.

(1975)

(1972)

Sci.

Res.

(ed.

Bishop,

Nat.

A. J.

Ross,

J. M.,

Sci.

Virology

U. S. A.

Mol.

Biol.

P. D.

Boyer),

and

Goodman,

U. S. A.

65,

72,

Vol. 67, No. 3, 1975

13.

Levinson, J., Jackson, J.

Bishop, and

14.

Kacian, L.

15.

Collett,

16.

Garapin,

D. L., Grossman M.

Wang,

18.

Garrett,

19.

Fraenkel-Conrat,

20.

Barlow,

22.

Keller,

23.

Molling,

Verma,

J.

D. P.,

J.

(1973)

J.

and

Pitot,

B.,

and

A. P. (1966) and Davies,

Gerard,

G. F.,

D.,

Viral.

16,

W., 589-598. Viral.

12,

H. C.

in Procedures D. R.) pp.

Green,

N.,

Fanshier,

Analyt.

Biochem.

Virology

in 444-454.

Nucleic

(1973)

J.

14, Acid

Harper

Viral.

Res. and

10,

142. Crouch,

K., Bolognesi, Hausen, P. I.M.

(1975)

R.

(1971) J.

(1972)

Proc.

Nat.

Acad.

D. P., Bauer, H., Busen, W., Nature N. B. 234, 240-243. Viral.

15,

121-126.

955

L.,

1512-1521.

(1961)

M,

(eds., New York.

000-000.

(1973)

A.

L.,

Enzymology Press,

Quintrell,

Tsugita,

and

Fanshier,

in Methods in 150-173, Academic

P., Levinson, J. Viral. 6,

D. S.,

L.,

(1973)

(1975)

P. H.

, and Mathias, G. , Cantoni, New York.

W. , and 3360-3364.

and

McDonnell, J. M. (1970)

Singer,

Sullivan, 112-195.

29E,

A. J.

Wilkinson,

N., 42,

S.

Faras,

H.,

Grandgenett, 1136-l

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Quintrell, Virology

Duesberg,

C. T., 342-348.

J. (eds. Row,

24.

and

W., (1970)

Spiegelman, and K. Moldave)

S. , and

L.,

52,

21.

and

A. C., Bishop,

and 17.

BIOCHEMICAL

Sci.

U. S. A.

Plassmann,

69,

H-W.,

54-58.