The inhibition of DNA synthesis by calf-thymus polymerase by X-irradiation of the primer DNA

Vol. 5, No. 6,196l

BIOCHEMICAL

THE INHIBITION

AND BIOPHYSICAL RESEARCH COMMUNlCAflONS

OF DNA SYNTHESIS BY CALF-THYMUSPOLYMEZASE

BY X-IRRADIATION OF THE PRIMER DNA.' K.A. Department

of Radiology,

Stacey*

Yale University

School of Medicine,

New Haven, Corm., U.S.A.

Received

July

bong in cells,

‘25, 1961

the many effects

one of the most characteristic

DNA synthesis, and its

produced by ionizing

whether temporary

exceptions

Two obvious

possible

is the inhibition

or permanent.

has recently

been reviewed

explanations

radiations

of this

This statement by Gouthier

effect

to DNA synthesis

DNA is damaged in such a way that

it

Bollum,

Anderegg,

rat-liver X-irradiation. to test

in tissues

fully

synthesize

(1960)

the first of

that regenerating

thymidylic

of the experiments

possibility,

for

from the experiments

in which Di?A synthesis

The object

sn alternative

X-irradiation 1

McElya and Potter

csn still

polymerase

seems likely

or that the

cannot be replicated.

Though Okada and Hempelman (1959) found evidence it

(1961).

are that the

block occurs in enzymes essential

of these hypotheses,

of

kinase and DNA is blocked by reported

here was

that DNA damaged by

may not be so easily

replicated

by the DNA

This work was supported by research grants A-3634 and E-2920 from the United States Public Health Service to Dr. P. BowardFlanders. Permanent address: Medical Research Council, Experimental Radiopathology Research Unit, Hammersmith Hospital, Ducane Road, London, W. 12. 486

Vol.5,No.6,1961

synthetic

BlOCHEMlCAl

mechanism.

of X-irradiated

Preliminary

DNA to "prime"

preparations

from calf

but only by relatively an extract

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

results the action

thymus (Bollum, high doses.

from regenerating

show that

of polymerase is indeed reduced,

1960)

Similar

rat-liver

the ability

experiments,

using

have been reported

by

Wheeler end Okada (1961). METHODS The method described purification followed

of DflA polymerase exactly

preparation.

thymus glands was

to the end of the third

stage, his Fraction

incorporated

a suggestion

activated

mercaptoethanol. and thymidine Canellakis. nucleoside

activity

The radioactive were prepared

were obtained

primers X-rays

5'-triphosphates

in the laboratory polyphosphoric

with a preparation

rich

the acid insoluble

was found of deoxgcytidine

of Dr. E.S. the

acid (Chambers, 1959). to the

by column chromatography (Canellskis, The non-radioactive triphosphates The rapid

technique

using

by Bollum (1959) was used to radioactivity.

weight 4.5 x IO6 by light

Calf

scattering)

method was used for the primer. were produced by delivering

at a dose rate

on

in kinases made from E. coli

from Pabst Laboratories.

paper discs devised

detergent

it

the monophosphates were raised

Gottesman and Kammen, 1960).

(molecular

Acting

per hour.

the system somewhat more than

and these were then purified

determine

was 3 *M of labelled

per mg. of protein

with radioactive

isolation,

C,

with this

The mOnOphOSphateS were made by incubating

triphOSphateS

filter

here were performed

by Dr. J. Krakow of Yale University,

cysteine

After

described

The specific

nucleotide

for the partial

from calf

and the experiments

that

by Bollum (1960)

487

prepared

by the

The irradiated

varying

of 3,750 rads/min.

thymus DNA,

doses of 250 kVp

to 0.4% solutions

of

Vol. 5, No. 6,196l

DNA, which bath

for

BIOCHEMICAL

were then 10 mins.

absolute it

to 0.2%,

and chilled for

manifested

confirmed;

protein,

2 pmoles

each of dCTP,

200 pg.

cysteine,

2.5

dTTP,

dATP,

with

P32 such that

per micromole.

In

each experiment

into

the

acid

The almost

this

enzyme into

DHA

was

unheated

DNA.

DNA, 100 kg.

of

of Mg++ and 7.5 or 30 wrnolf% either

the

the

activity

the

insoluble

water-

of the

of heated

pmoles

with

a boiling

en ice-bath.

was incorporated

labelled

and dCHP32

in

of denaturation

to prime

contained

dGTP,

in

some form

a capacity

mixture

heated

rapidly

no radioactivity

The reaction

d-TTP

diluted

requirement

before

AND’ BIOPHYSICAL RESEARCH COMMUNICATIONS

dCTP or the was about

incorporation

material

IO6 c.p.m.

of dTMP32

was measured

in

parallel.

RESULTS AiTD DISCUSSION The time DHA, under using,

course

the

of the

conditions

as primer,

described

DNA that From this,

of X-irradiation.

incorporation

of radioactivity

above,

had been

is

subjected

intO

shown in Fig. to various

1

doses

end Table 1 it can be seen that the

synthesis of new material is strongly inhibited

by large doses

of X-rays and that within the experimental error there is no great difference

in the amounts of incorporation

of d-TMP and d-CMPat any

dose level so far examined. This contrasts strongly with the results

obtained from

by Ckada

(1960)

regenerating

striking

rat

change

in (both

X-irradiation

of the

the

findings

decline in

the

with rat-liver

liver

the

incorporation

who, using

ratio

and Potter,

of thymidine

tritium-labelled) primer.

of 'Jheeler dose

(Bollum

in

These

priming

results (1961)

efficiency

system. 488

preparation

1958),

found

a

to cytidine as the

and Okada the

an unfractionated

result

of

do however who found

support

a similar

of irradiated

DUA,

Vol.5,No.6,1961

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table The fraction

of surviving

of incorporation of incorporation

amt.

amt.

1

priming

at 10 mins. at IO mins.

Dose (rads) 7,500

ability

using using

eqxessed

irradiated control

as

DXA primer DIG primer

dCPd2

dTMP32

128

105

;0,000

90

75

94 ,000

32

34

14

19.6

200,000

10

(mitt) FIG.

x ,00

,o

(min) la

FIG.1 b

n of the label&d The time-couree of the incorporat compound (Fig. la dCMP32, Fig. lb. TIC $3 ) in an acid insoluble form, using the conditions described in the text and primer irradiated with these doses of X-rays, xcontro1.U 7,500 rads, 04 20,000 rads, 6;-----h 94,000 rade,&-A200,OOO rads. TIhese results newly

synthesized

are preliminary, was only

a small 489

for fraction

the

total (-I$)

amount

of D2A

of the

amount

10

Vol.5,No.6,1961

BIOCHEMICAL

of DNA used as a primer.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Since the addition

at the end of the normal incubation amount of synthesis low activity

of the enzyme used here.

priming

ability

system.

Bollum (1960b)

of DNA was not abolished

would not anticipate damaged regions

total

inhibition

are distributed

reactivation

and those of Okada assaying results

the effects obtained

polymerase

of irradiation

preparations

is apparent

rads,

but one

because the

along the DNA molecules, primers.

bacteriophage

(Harm,

synthesis

of

between these results

emphasizes that

and experiments

100,000

of the partial

The difference

(1960)

it

has remarked that the

be effective

1958) does suggest the possibility DNA damaged by X-rays.

is due to the

of synthesis

of X-irradiated

a comparable

of DNA to stimulate by

randomly

@nd the undamaged ends may still Multiplicity

However,

does reduce the ability

in this

substrate

time stimulates

the low degree of conversion

that X-irradiation synthesis

of fresh

the system used for

may greatly

modify

with more highly

are therefore

the

purified

in hand.

ACKNOWLEDGEMENTS It is a pleasure encouragement received

to acknowledge

the constant

of Dr. P. Howard-Flanders,

from Dr. E.S. Canellakis

the great

and the technical

advice

and

help I assistance

of Miss Eva Simson. REFERENCES Bollum, F.J. and Potter, V.R. J. biol. Chem. Z!& 478 (1958). Bollum, F.J., Anderegg, J.W., McElya, A.B. and Potter, V.R. Cancer Res. 20, 138 (1960). Bollum, P.J. J.Tiol. Chem. 2 2733 i1959). Bollum; F.J. p. 163. "The CeIP I;ucleus" ed. MLLxhell, Academic Press, N.Y. Canellakis, E.S., Gottesman, M.E. and Kammen, H.O. Biochim. Biophys. Acta. , 82 (1% Chambers, R.W. J.A.9 .S. l 31, J% (1959) Gouthier, R. Progre, 88 in'8iophysics. i, 3, (1961). Okada, S. Nature. *2, ;9;, (1960). Okada, S. and Hempex 9 . . Int. J. Rad. Biol. 1, 305, (1959 ). Wheeler, C.M. and Okada; S. Int. J. Rad. Biol. 2,?3, , (1961). 490