Presence of poly (rA): poly (dT)-dependent DNA polymerase in mouse myeloma cells

BIOCHEMICAL

Vol. 46, No. 5, 1972

AND BlOPHYSldAL

PRESENCE OF POLY(rA)

RESEARCH COMMUNICATIONS

:POLY(dT)-DEPENDENT

DNA

POLYMERASE IN MOUSE MYELOMA CELLS

Iwao Suzuka

and Masahiro

Muto

Department of Biophysics National Institute of Animal Health Kodaira, Tokyo 187, Japan

Received February 2, 1972 Summary. An RNA-dependent DNA polymerase analogous to that of normal cells has been found in mouse myeloma cells. This enzyme, which is activated by Mn2+ ion, specifically copies the poly A strand of poly(rA):poly(dT) hybrid to synthesize dTMP homopolymer.

RNA-dependent all

oncogenic

RNA viruses(l-3),

even in normal typical to

DNA polymerase

cell

cells(5-10).

which

synthesize

It

flow

information

synthesis

in myeloma cells.

the

evidence

and Methods.

saline

0.05 M Tris-HCl,

in the

of

known as a

interest

to

&-globulin

paper,

presence

5) and

we have

shown

which

of

hybrid. Tumor cell

was maintained

in C3H mouse. Solid buffered

for

in

and continues

was therefore

In the present

dTMP homopolymer

myeloma protein

are well

of an enzyme in mouse myeloma cells

poly(rA):poly(dT) Materials

genetic

cells(4,

potential

r-globulin(l1).

the

synthesizes

in transformed

has proliferative

of

has been detected

Myeloma cells

elucidate

first

activity

tumor(about

and homogenized pH 7.8,

0.05

X5563,

by subcutaneous

forming

IgG

transplantation

4 g) was washed in phosphatein 4 ml of M KCl, 1874

Copyright01972,byAcademicPress,Inc.

line,

a buffer

containing

1 mM DTT(dithiothreito1)

BIOCHEMICAL

Vol. 46, No. 5, 1972

and 1 mM MnCl2(buffer for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The homogenate

A).

1 min and centrifuged

at 20,000

was suspended in 5 ml of buffer 37'

in the presence

After

obtained

from the

centrifuged

at

150,000

was fractionally

rated

ammonium sulfate

A and incubated

xg for

30 min.

precipitated solution

was retained,

the

Co.). to that

and the mixture

containing

was

xg super-

addition

of

satu-

0.05 M Tris-HCl,

2O);

the

pH

0.3 to 0.77

in 4 ml of buffer

, and dialyzed

at 4O. Protein

Chemical

The 150,000

dissolved

20% qlycerol(v/v)

same buffer

Lowry

then

15 min at

was combined

by the

at

at 4O

The pellet

for

P-4O(Shell

xq centrifugation,

8.1 and 0.1 mM Na-EDTA(saturated

containing

15 min.

the supernatant

20,000

natant

fraction

xg for

of 0.4% Nonidet

the centrifuqation,

was sonicated

overnight

was determined

A 500 ml

of

by the method of

et -- a1.(12). The DNA polymerase

of the

activity

assay procedure

The standard in final

reported

assay mixture

volume

of

was measured by a modification by Stavrianopoules

contained

0.1 ml:

the

(102 cpm/pmol); protein.

out

for

of each reaction radioactivity poly(dT),

mixture

was determined

from Miles

[3H]dATP(12.9

poly

C/mmole)

Amersham.

Results

and Discussion.

After

the

reaction,

0.08

ml

acid-insoluble

and poly

[3H]dTTP(ll.3

1875

t3H]dATP

the enzyme

to Bollum(l3).

d(A-T)

actively

or

of

Poly(rA)

A were C/mmole)

and

from Radiochemical

As shown in Table

from myeloma cells

t3H]dTTP

addition

and the

were purchased

0.12 M Xl;

and 10 rg of enzyme

according

Laboratories.

Centre,

preparation

37'.

was taken

poly(rA):poly(rU),

acquired

of

was begun by the 90 min at

components

pH 8.1;

2 pq of poly(rA):poly(dT)

The reaction

and carried

following

0.05 M Tris-HCl,

2 mM DTT; 1 mM MnC12; 5.2 nmoles either

et -- a1.(7).

1, a crude

catalyzed

the

enzyme incorpo-

:

Vol. 46, No. 5, 1972

Table

BIOCHEMICAL

1. Polymerase activity presence of various

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of mouse myeloma cells templates

Template Exp.

1.

pmoles dTMP

in the

incorporated dAMP

None Poly(rA):poly(dT) Poly(rA) :poly(rU) P0iy d(~-T)* Poly A

1 1 238 6 1 15 2 Exp. 2** None 1 Myeloma DNA 29 RNA Myeloma cytoplasmic 19 The reaction mixture was the same as in Method. Myeloma DNA and myeloma cytoplasmic RNA were prepared according to Marmur(14) and Muto and Morita(15). *, 50 nmoles of dATP were added to the reaction mixture. **, 50 nmoles of each dATP, dCTP and dGTP were added to the reaction mixture.

ration

of dTMP into

poly(rA)

:poly(dT)

proportion

polymer hybrid.

only

in the presence

The incorporation

to the concentration

of

this

of

increased hybrid(Fig.

almost 1).

Under

230

0 3

1 )I6

Fig.

2

3

I

4

~LV(RA).POLV(DT)

l(left).

Poly(rA):poly(dT)-dependent DNA polymerase as a function of template concentration. The reaction was carried out as described in Method except that poly(rA): poly(dT) was added as indicated in the figure. Fig. 2(right). Poly(rA):poly(dT)-dependent DNA polymerase aCtiVlty as a function of divalent catlon concentration. The assay was the same as in Method except that [MnL+] or [Mg2+] was added as indicated in the figure. activity

1876

in

Vol. 46, No. 5, 1972

the

BIOCHEMICAL

same conditions,

observed.

On the

plasmic the

no significant other

in the presence

d(A-T),

is

zation

of dTMP catalyzed

incorporation

and poly

These results

hybrid

the most effective

of dAMP

was

was found

myeloma DNA, or myeloma cyto-

RNA. Poly(rA):poly(rU)

and that

incorporation

a slight

hand,

of poly

incorporation.

this

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A were inactive

indicate

template

that

for

poly(rA):poly(dT)

in promoting

polymeri-

by the enzyme from mouse myeloma cells

the enzyme exclusively

transcribes

the

ribo

strand

of

hybrid. The divalent

cation

requirement

dTMP guided

by poly(rA):poly(dT)

corporation

was markedly

mM Mn2+. only

In contrast,

35% of that

extensive

reduction

the polymerization

shown in Fig. by the

incorporation

obtained

concentration

is

stimulated

the

of higher

for

2. The in-

addition

with

of

optimal

Mn2+ . Furthermore,

with

of Mn2+ or Mg2+ resulted of enzymatic

activity

to

of

0.25-2

Mg2+ was

the presence in an

synthesize

the

was sharply

de-

polymer. As shown in Fig.

3A,

the incorporation

pendent

upon the enzyme concentration

from 2 to 10 pg of

protein

per assay.

of enzyme protein

abolished That

its

The heat

ability

the polymerized

by lack

to synthesize

was DNA-like

of RNase degradation,

susceptibility

and alkaline

of the

incorporation(Curve remained

concentration with

long

resistance.

incubation

reached

for

gradually.

at only

the

was supported

rate

course

of polymer-

90 min when low enzyme however,

dropped

By increasing 2 of Fig.

30 min,

1877

3A).

to DNase degra-

3B),

at least

4 fold(Curve

a plateau

product

The incorporation,

times

of Fig.

As shown by the time

1 of Fig.

constant

was used.

enzyme concentration ration

dTMP polymer(x

product

dation

ization

treatment

3B),

followed

the the

incorpo-

by a rapid

Vol. 46, No. 5, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 0

2.5 5 7.5 pb PROTElN

10

0

60

120

180

TINE hlN)

Fig. 3A. Poly(rA):poly(dT)-dependent DNA polymerase activity as a function of enzyme concentration. The assay was the same as in Method except that enzyme protein was added as indicated in the figure and the reaction time was 120 min. x, The enzyme protein was treated at 600 for 5 min. B. Poly(rA):poly(dT)dependent polymerase activity as a function of reaction time and effect of enzyme concentration. The assay was the same as in Method except that total reaction mixture was 0.4 ml. A 0.06 ml aliquot was taken at each time interval as indicated in the figure. Curve 1, 40 pg of enzyme protein were added at 0 time; Curve 2, 160 pg of enzyme protein were added at 0 time: Curve 3, 120 pg of enzyme protein were added at 45 min under the conditions of Curve 1; Curve 4, 120 pg of enzyme protein were added at 120 min under the conditions of Curve 1.

decrease

of

when the

additional

3B).

the

incorporation.

In contrast,

when the

the

45 min product

observed

the

and only

4 of Fig. was not

min),

3B).

relatively

behaves

a DNA:RNA hybrid

while

ceptible

product.

to DNase present

form,

Therefore,

the

latter the

at in-

place

experiment, to DNase

first

product synthesis

product

in the enzyme preparation.

1878

took

susceptible that

to a DNA-like

3 of Fig.

of the

In a preliminary

These data may suggest

leads

increase

a decrease

degradation. like

was observed

enzyme was added additionally

time(l20

was not

immediately(Curve

fashion

enzyme was added at 45 min(Curve

maximum incorporation corporation

Similar

was susSimilar

Vol. 46, No. 5, 1972

hybrid

BIOCHEMICAL

product

E. coli

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

has been detected

DNA polymerase

in the presence

In summary, the data an enzyme isolated

in the product

described

enzyme which

is guided

template.

The presence

of

system to

study

a mechanism for

of myeloma cells.

Recently,

dependent

DNA polymerase

of active

proliferating

myeloma polymerase the

by ribo

reverse with

it plays

poly(rA):poly(dT)

polymerases

from RNA tumor viruses

hybrid(4,

by Mg2+ (4, whether

18,

19).

cells(5,

of

Studies

of DNA:RNA hybrid

that

RNA-

The specificity

of

stimulation

On the other

duplexes,

are currently

activity

hand,

the cells

such as poly(rA): is

in progress

can be directed

by

DNA

and virus-transformed

and their

the myeloma polymerase

information

and the

7-9).

all-rib0

5, 18-20),

of

as useful

of RNA-dependent

from normal

poly(rU)

of the hybrid

in the gene amplification

17).

characteristics

to the presence

a hybrid-

strand

flow

that

the gene amplification

a role

cells(l6, for

is

indicate

has been suggested

polymerases

respond

here probably

by

RNA(6).

such an enzyme may serve

from RNA to DNA and a relation

Mn2+ resemble

of ribosomal

from mouse myeloma cells

dependent

polymerized

facilitated to see

by RNA strand

from myeloma cells.

References. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

D. Baltimore, Nature 226(1970) 1209. H. M. Temin and S. Mizutani, Nature 226(1970) 1211. s. Spiegelman, A. Burny, M. R. Dass,x Keydar, J. Schlom, M. Tranicek and K. Watson, Nature 227(1970) 563. R. C. Gallo, S. S. Yang and R. C. mg, Nature 228(1970) 927. E. M. Scolnick, S. A. Aaronson, G. U. Todaro and W. P. Parks, Nature 7229(1970) 318. L. F. Cavalier1 and E. Carroll, Biochem. Biophys. Res. Commun. 41(1971) 1055. J. G. Stzrianopoulos, J. D. Karkas and E. Chargaff, Proc. Natl. Acad. Sci. U. S. 68(1971) 2207. J. C. C. Maia, F. Rouge?% and F. Chapeville, FEBS Letters 18(1971) 130. p. E. Penner, L. H. Cohen and L. A. Loeb, Nature 232 (1971 58. H. B. Bosmann, FEBS Letters -19(1971) 27. 1879

Vol. 46, No. 5, 1972

11. 12. 13. 14.

15. 16. 17. 18. 19. 20.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

D. Nathans, J. L. Fahey and M. Potter, J. Exptl. Med. 108 (1958) 121. 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. S. Randall, J. Biol. Chem. 193(1951) 265. F. J. Bollum, in: Methodsn enzymology Vol. 12 Part B, eds. L. Grossman and K. Moldave(Academic Press, New York, 1968) p. 169. J. Marmur, J. Mol. Biol. A(1961) 208. M. Muto and T. Morita, Gann(Japan) 62(1971) 107. M. Crippa and G. P. Tocchini-ValentGi, Proc. Natl. Acad. Sci. U. S. 68(1971) 2769. A. Ficq and?. Brachet, Proc. Natl. Acad. Sci. U. S. -68 (1971) 2774. E. Scolnick, E. Rands, S. A. Aaronson and G. J. Todaro, Proc. Natl. Acad. Sci. U. S. 67(1970) 1789. N. C. Goodman and S. Spiegelman , Proc. Natl. Acad. Sci. U. S. 68(1971) 2203. S. Spiegelman, A. Burny, M. R. Das, J. Keydar, J. Schlom, M. Tranvicek and K. Watson, Nature 228(1970) 430.

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