Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

8lOCHEMlCAL

Vol. 99, No. 4,198l April

AND BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

30, 1981

POLYAMINES STIMULATE

1361-1368

NATURAL RNA-DIRECTED DNA SYNTHESIS BY RAUSCHER

MURINE LEUKEMIA VIRUS DNA POLYMERASE

1

Received

Stuart

L. Marcus '*,

Memorial

Sloan-Kettering

'Haskins

Laboratories

March

Steven

W. Smith',

Cancer

and Cyrus

Center,

J. Bacchi'

New York

of Pace University,

NY 10021,

New York,

and

NY 10038

18,1981

SUMMARY. In the presence of optimal concentrations of Mg2+, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem$ate-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70s RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis. The relative

inefficiency

mammalian

type

synthetic

template-primers

viral

C retroviral

factors

other

ency of --in vivo cations,

the

been shown systems

and activated

than

reverse

polymerase

both

We report Rauscher

DNA synthesis

by biologically

the stimulatory

*

globin

To whom correspondence

effecters

murine

for

active

for

the

(R-MuLV)

polyamines.

that

or

the effici-

the

organic

since

in various

stimulation

they

have

enzyme

of natural

DNA polymerase-catalyzed

Analysis

spermine

of

cellular

optimizing

in vitro -___

time

by isolated

utilization

that

to investigate

as a template-primer

suggested should

virus

their

of cDNA synthesis

the first

leukemia

mRNA.(dT),2-,B

polyamine

be required

RNA and DNA synthesis here

with suggests

We chose

as possible

to stimulate

rabbit

might

DNA synthesis

compared DNA (l-3)

transcription.

polyamines,

(4-11).

RNA-directed

DNA polymerases

RNA-directed,

using

of natural

of such stimulation complex

acted

and spermine

to increase

as

the affinity

be addressed 0006-291X/81/081361-08$01.00/0 1361

All

Copwrghr 7 1981 r&h/r of reproduc,lion

by Academrc Press, Im,. rn aqy form reserved.

Vol. 99, No. 4,198l

8lOCHEMlCAL

of R-MuLV DNA polymerase decrease

the quantity

synthesis

for

of primer

on a fixed

quantity

R-MuLV 70s RNA-directed inhibitory

in the

to stimulate

absence

molecules

RESEARCH

complex

required

of globin

for

only

optimal

rates

primer

suggest

as to

of cDNA

stimulated

at RNA concentrations

primarily

at the

as well

Spermine

Our results

DNA synthesis

COMMUNICATIONS

lo-fold

mRNA template.

of polyamine.

of cDNA synthesis

MATERIALS

BIOPHYSICAL

template-primer

cDNA synthesis

RNA-directed

of initiation

the

AND

which that

were

spermine

by increasing

the

acts

efficiency

terminus.

AND METHODS

Reagents: (3H)-dTTP and (3H)-dGTP were purchased from the New England Nuclear Co. Unlabeled dNTPs were obtained from P-L Biochemicals, Inc., as was Rabbit globin mRNA was the kind gift of Dr. J. Vournakis of Syracuse (dT)12-18. University. Polyamines as HCl salts were obtained from Sigma Chemicals Co. Purified R-MuLV and avian myeloblastosis virus (AMV) were provided by Dr. J. Cole of the NCI. R-MuLV DNA polymerase was purified as previously described (2). Genomic 70s RNA from R-MuLV or AMV was purified after extraction by velocity sedimentation (1). Rabbit globin mRNA was annealed to (dT)l2-18 at a weight ratio of lO:l, respectively, in 0.05M Tris (pH 7.8) buffer (2). DNA polymerase assays: Reactions were carried out in a total volume of 100 ~1 and contained 50mM Tris-HCl, pH 7.8, 1mM dithiothreitol, 10 ug of bovine serum albumin, and 1mM Mg2+ (as MgC12). Unlabelled dNTPs were present at individual concentrations of 0.2mM and labelled substrate at 10pM. Unless otherwise stated, 1 vg of template-primer complex was used per assay, and reactions were incubated at 370 for 1 hour. Trichloroacetic acid-insoluble radioactivity was then collected on glass fiber filters and quantitated as previously described (2). RESULTS Genomic above, fold

70s RNA-directed

the addition

of substrate

spermine

the stimulation addition

of the

and absence

(Fig.

at RNA concentrations

sence appears

of polyamine. largely

1) revealed

~100 ng per

The stimulation

to be due to negation

to control

assay,

which

were

of 70s RNA-directed of the

1362

inhibition

assays.

The

of 200-500uM,

while

mixtures

stimulation

in >lO-

only

did

not not

RNA concentrations that

shown

provided

up to 2mM (data

to increasing

resulted

addition

concentration

at concentrations response

of spermine

Spermidine

to reaction

conditions

reactions

as compared

at an optimal

or putrescine

DNA polymerase

the reaction

to 70s RNA-directed

was 50uM.

of spermine,

cDNA synthesis,

Under

incorporation

concentration

of cadaverine

stimulate

only

of spetmine

stimulation

optimal

DNA synthesis.

shown).

by spermine

reactions

the

significantly

in the

inhibitory

half

Analysis presence occured

in

the ab-

therefore

of cDNA synthesis

caused

8lOCHEMlCAL

Vol. 99, No. 4,198l

Fig.

1.

Effect of increasing concentrations catalyzed by R-MuLV DNA polymerase Incubation was of 50~M spermine.

by high

RNA concentrations.

"nicks"

observed

productive (Fig.

enzyme binding

degree

Spermine

of interpreting

sites

of uncertain

and for annealed

that

spermine

than

the

in which which

exogenous

to (dT),2-,8

of the

kinetics

as the quantity

led

of

primer

size

analysis

revealed

no difference

finally

in size

samples

and cDNA synthesized

Kinetic

analysis

of the

both

effect

as nonreaction

of cDNA synthesis

cDNA synthesis. internal

a system

could

to

>lO-fold

2B) showed the

initial

were

between

in the presence

of sperm ine with

by spermine,

and analysis

of the

significantly

1363

(data

(250~iM)

and sub-

reaction

as well

increased. incubation

cDNA synthesized

respect

mRNA

complex

at optimal

of spermine

globin

template-primer

15 and 120-minute

distribution

was minimal,

namely

velocity

synthesized cDNA from

that

binding

RNA-directed

isolation

be supplied,

The diffi-

enzyme

of natural

on this

stimulated (Fig.

of globin

rate

70s RNA containing

DNA synthesis

concentrations

However,

initial

mRNA-directed

molecules

the reaction

may serve

70s RNA-directed

of RNA damage during

was also

of product

which

of

of such synthesis.

us to examine

(13-15).

R-MuLV polymerase

spermine

using

virus, of the

the

of globin

the possibility

by the

optimal

stimulated

results number

frozen

Kinetics

extent

COMMUNICATIONS

may be due to the presence

from

(12).

final

as an effector

culty

synthesis

sites

RESEARCH

of genomic 705 RIIA on DNA synthesis in the presence (a) and absence (0) carried out for 30 min. at 370.

Such inhibition

in 70s RNA prepared

2A) showed

a greater

AND BIOPHYSICAL

not

periods in control

shown).

to vary ing mRNA.(dT)12-,8

8lOCHEMlCAL

Vol. 99, No. 4,198l

AND

BIOPHYSICAL

RESEARCH

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^ ‘0 - 14 x B l2 E 10 E

b a z s6

52

4

*2 -7 Liz -- 0

Fig.

2:

Effect of spermine on the kinetics of DNA synthesis directed by genomic 70s RNA with endogenous primers (A) and rabbit globin mRNA annealed to At all time points gel analysis of cDNA (dT)l2-18 as a primer (B). products revealed no significant quantity of double-stranded DNA synthesis, and the presence of 100 ug/ml actinomycin D in reaction mixtures did not affect the kinetics of DNA synthesis (data not shown).

concentrations

is presented,

The apparent that

in

of

the

mine

in the

Km for

template-primer

the presence

of 25, 50,

reaction

also

appeared

form of a double-reciprocal

plot,

in the absence

was 5.6 ug, while

or 250uM spermine

to

increase

of polyamine decreased

significantly,

to 0.56 but

only

in

Fig.

vg. at

3.

The Vmax

higher

sper-

concentrations. By making

molecules,

use of our

ability

stimulation

initiation

of cDNA synthesis.

to

quantity

a fixed determined

the absence

(250 in

the

of spennine,

to vary

we were

as (dT)12-,8,

nism of polyamine

sis

60 90 120 150 180 210 Minutes at37"

30

ng)

presence

a

able

might

to determine

globin of

various

and

spermine

primer-to-template

of the

1364

(dT)12-,8

initial concentrations

weight

primer

an additional of priming

quantities mRNA

of potential

whether

be the stabilization

Increasing of

the concentration

ratio

rates

mecha-

sites, were of (Fig.

for

annealed cDNA

synthe-

4).

of 10 was required

In

BIOCHEMICAL

Vol. 99, No. 4,1981

AND

pg (globm

Fig.

to

produce

quantities

3.

template

ratio

mRNA + dTlz-lx)

RESEARCH

COMMUNICATIONS

-/

Effect of spermine on the apparent Km and Vmax of the reaction of R-MuLV DNA polymerase-catalysed DNA synthesis with respect to varying globin mRNA.(dT)l2-18 template-primer complex concentration. Final concentrations of the template-primer complex were 0.25, 0.5, 1, 2.5, and 5 vg/lOO ~1. Spetmine was present at a concentration of 0 (o), Results are presented in the form of 25 (o), 50 (A), or 100 (n) PM. a double reciprocal plot.

optimal of

BIOPHYSICAL

rates

spermine was

of present

25-fold

but

synthesis, until lower

at than

this

250nM that

ratio

decreased

spermine

required

in

with

increasing

the

optimal

primer-to-

the

absence

of

spermine.

ng WV,?-,x

Fig.

4.

Effect of spermine on the rate of globin mRNA-directed DNA synthesis in the presence of changing concentrations of (dT)l2-18. Rabbit globin mRNA was present in each assay at a fixed quantity of 250 ng. Incubation was carried out for 30 minutes at 370. Spermine was present at final concentrations of 0 (o), 50 (o), 100 (A), and 250 (A) PM.

1365

Vol. 59, No. 4,19Bl

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Spermme (mM) Fig.

5.

Effect results

Effect of spermine on rates disruoted R-MuLV. Reaction P-40,'which provided optimal total virus used per assay. hour. Control cpm incorporated

of spermine

seen

in the

spermine

caused

synthesis

(Fig.

on endogenous

reconstituted

significant 5),

(2).

as low as 5pM (data

not

synthesis,

at higher

This

inhibition

since

the

(data

not

system

did

(Fig.

l),

out

Inhibition

R-MuLV

at pre-determined was observed

Spermidine

addition

concentrations

than

also

affect

of NP-40 the

caused (data

of polyamine used

of

RNA-directed optimal

spermine

to the

concentrations

at spermine

to be due to interaction

not

In contrast

increasing

of endogenous

of the same quantity

to a reconstituted spermine

shown).

appeared

addition

system

was carried

and Mg2+ concentrations

although

R-MuLV cDNA synthesis.

inhibition

which

of endogenous DNA synthesis by detergentmixtures contained 0.015% (v/v) Nonidet rates of DNA synthesis with.the 2 ug of Incubation at 370 was carried out for 1 = 3,620.

cDNA

detergent concentrations

inhibition not

of DNA

shown).

with

detergent

in the endogenous

previously

observed

reaction

stimulation

by

not shown).

DISCUSSION We have shown mines

spermine

synthesis

at optimal

and spermidine

catalyzed

70s RNA-directed

that,

stimulate

by R-MuLV reaction

divalent

cation

70s RNA and globin

DNA polymerase.

is most

evident

concentrations,

at

1366

the polya-

mRNA-directed

cDNA

that

stimulation

of the

RNA concentrations

which

The fact

inhibit

BIOCHEMICAL

Vol. 99, No. 4,198l synthesis

in the

converting

absence

of spermine

non-productive

mechanism

recently

enzyme

suggested

DNA-directed

DNA synthesis

The addition

of spermine

R-MuLV enzyme for lo-fold,

which

reaction (Fig.

4).

achieved absence

(Fig.

mixtures.

nucleic

polyamine

stabilization

creased

acid

efficiency

Our finding thetic

reactions

(Fig.

5) differs

Virion (20)

It

is

RNA bound already

addition

examining synthesis. functional

from in

the effects This role

approach played

obtained

were in

the

effects

of

the salt

con-

(as ammonium sulfate) effects

ability

of

we have

of spermine

specifically

to this

complex

to endogenous

noted

to

study,

resulting

to

in in-

in a reconstituted structure

cation

native

protein

may provide core

for

the

this

priming block

possibility

structural

and polyamine a greater proteins

1367

proteins addition

understanding in the

DNA synDNA synthesis

reaction

containing

may therefore

examining

purified,

RNA-directed

R-MuLV inhibits

configuration

organic

by virion

by (dT)12-18

by increasing

reviews)

by detergent-disrupted

of such

mRNA-directed

of DNA synthesis.

currently

with

for

and,

at limiting

required

the stimulatory

(16).

approximately

globin

those

the stimulatory

for

a ribonucleoprotein

We are

structures

that

of spermine

results

of an exogenous

than

that

a

of the

of cDNA synthesis

enzyme-template-primer

addition

catalysed

rates

concentration

17-19

affinity complex

the

by

of activated

we observed

less

structure

of initiation

be in an optimal

DNA synthesis. ion core

secondary of the

that

optimal

probable

sites,

of cDNA synthesis

substituted

(see

the

stimulated

Z-fold

may act

DNA polymerases

template-primer

also

ammonium ion

to the well-documented

stabilize

C retroviral

of priming

be even partially

spermine

stimulation

the stimulation

Spermine

COMMUNICATIONS

initiation

increased

must be emphasized

or the

to useful

type

of spermine,

It

(as KCl)

reaction

relate

3).

that

the polyamine

for

concentrations

not

sites

mixtures

the efficiency

at (dT)12-18

centration the

accounts

RESEARCH

1) suggests

mRNA.(dT)12-18

In the presence

could

binding

by mammalian

partially

of spermine.

spermine

(Fig.

to reaction

by increasing

BIOPHYSICAL

to explain

the globin

mRNA concentrations

AND

process

viral

(Fig.

1, 2A).

core

proteinsmay

of cDNA synthesis. sites

used for

The priming

by reconstituting (21)

vir-

and RNA and

on RNA-directed of the

DNA

possible

of proviral

synthesis

Vol. 99, No. 4,19Bl

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGEMENTS The authors express there sincere thanks to Dr. John Vournakis of University for his generous gift of rabbit globin mRNA, to Dr. J. Cole for purified preparations of R-MuLV and AMV, and to Dr. M. J. Modak of Institute for general counsel. We thank Dr. N. Sarkar for his support agement. This work was supported, in part, by NC1 grants CA 18369 and

Syracuse and Gruber this and encourCA 08748.

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

2: 16. 1':: 19.

20. 21.

Marcus, S.L., Sarkar, N.H., and Modak, M.J. (1976) Virology 2, 242-254. Modak, M.J., and Marcus, S.L. (1977) J. Biol. Chem. 252, 11-19. Marcus, S.L., Sarkar, N.H. and Modak,-M.mgTBiochim. Biophys. Acta 519, 317-330. Abraham, K.A. (1968) Eur. J. Biochem. !j, 143-146. Wickner, W., Schekman,.,-Gem. and Kornberg, A. (1973). Proc. m. Acad. Sci. USA 70, 1764-1767. Janne,, -Bardin,'--- C.W. and Jacob, S.W. (1975) Biochemistry 14, 3589-3597. Yoshida, S., Masaki, S. and Ando, T. (1976). J. Biochem. 79, 895-901. Evans, J.A., and Deutscher, M.P. (1976) J. -BioT. -Chem. 251, 6646-6652. Barbiroli, B., Masotti, W., Moruzzi, M.S,, Monti, M.G., and Moruzzi. G. (1978) in w. in-Polyamine &. (R:A. Campbell,?Ed.) Vol. 1 pp. 217-229. Raven Fess. New York. Fisher, P.A.*and Korn, D-,(1979) J. Biol. Chem. 254, 11033-11039. Marcus, S.L., Lipshik, G., TruebaT G. and Bacchi3.J. (1980) Biochem. Biopti;;th~.WCom;;rncus~,S :027-1035 Sen, G., and Sarkar, N.H. (1979). Proc. m. 173&1740: -Acad. -*Sci' -USA"76-3 Kacian, D.L., Spiegelman, S., Bank, A., Terada, M., Metafora, S., Dow, L. and Marks, P.A. (1972) Nature New Biol. 235, 167-169. Ross, J., Aviv, H. and Leder,1972) Proc. Natl. Acad, Sci. -USA -3 69 264-268 Verma, I.M., Temple, G.F., Fan, H. and Baltimore, D.(1972rNature ___ -New -*Biol 235, 163-166. Smith, S.W. and Bacchi, C.J. (1981) J. Biol. Chem In Press Marcus, S.L., Cohen, S. (1971) Introduction to the Polyamines. Preme-m','NzKy. Tabor, C.W. and Tabor, H. (1976) Ann. Rev. Biochem. 45, 285-306. --7-Cohen, S.S. (1978) in --Adv. in Polyamine Res. -Campbell, Ed.) Vol. 1, pp. l-10. Raven Press, New York. Marcus, S.L., Smith, S.W., Racevskis, J. and Sarkar, N.H. (1978) Virology 86, 398-412. Marcus, S.L., Smith, S.W., Racevskis, J. and Sarkar, N.H. (1979) 2. m. m. 254, 4809-4813.

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