The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. coli DNA polymerase I

Vol. 61, No. 2, 1974

BIOCHEMICAL

THE EFFECT OF IONISING AND ACCURACY OF E.COLI

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RADIATION AND CHEMICAL METHYLATION UPON THE ACTIVITY DNA POLmERASE I

Roy Saffhill Paterson Laboratories, Manchester M20 9BX,

Received

October

Christie

Hospital

and Holt

Radium

Institute,

EnglamL 7,1974

SUMMARY The activity of E.coli DNA polymerase I decreases on treatment with Y-rays, In the case of the first two methylnitrosourea or dimethyl sulphate. agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an in vitro assay. There is no detectable change in the activity on treatment. ratio of DNA polymerase activity to 3' ->5’ exonuclease

INTRODUCTION DNA synthesis integrity

DNA polymerases to incorporate

several

recent

related

to cellular

error-prone

The high in the gens

should

of the DNA base

systems found

in vivo

from both

bacterial'

and mammalian

one wrong

in the activity treatment

with

sulphate

(DMS).

Abbreviations:

base

have suggested

that

has been

found

of the DNA polymerases of the agents

enzymes. upon E.coli

and accuracy the

methylating

MNlJ -

in several

0 1974 by Academic Pres.v, of reproductiorr in uny form

Inc. reserved.

Even in in vitro sources

have been

thousand.

in human leukaemic

possible

However,

to study

I and now wish

methylnitrosourea

DMS

the

-

in vitro

A mutant

small

effect to report b-irradiation

(MNU) or dimethyl

dimethyl

may be

6.

cells

investigating

enzyme following

methylnitrosourea,

the

in DNA synthesis c and tumour progression'.

DNA polymerase

agents

that

inaccuracies

We have been

of this

L

hundred

makes it

802 Copyright All right.r

in order

genome be maintained.

ageing" 74 , carcinogenesis

accuracy

and other

process

of the

DNA polymerase

fidelity

accurate

sequence

only

reports

be a very

sulphate.

changes

of carcinochanges or

Vol. 61, No. 2, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS AND METHODS DNA polymerases

and deoxyribonucleoside

the Boehringer prepared

by the method

purchased

from

in 0.02 rate

(London)

assium

rad/sec.

phosphate

The higher

pH 7.5

treatment,

buffer

have been given

untreated poly(dAdT)

template

This

was measured

were

identical

was taken

in every

phosphate

which

contained

50 nmoles

apart with

as poly(dA-dT).poly(dA-dT). from

To measure

total

j’

the base

labelled

DNA' under

of the

for

against

of the

assays

which

(total

volume

dGTP, 300 nmoles

[3H]-dTTP

assay

(as incubation. and when

[3H]-dGTP

was used.

with

3'-terminally

in the absence

of acid

O.jml)

nucleotide

was used

by incubation

standard

enzyme. tri-

was added before

The production

exonuclease

of the

two assays

of enzyme activity

enzyme)

was measured

The

deoxyribonucleoside

These

of

a poly(dA-dT).

accuracy

5nmoles

assay.

units

30 min.

using

3 x 107 cpm/assay

triphosphates.

as a measure

DMS) at 4'C.

to 0.02

using

2 x 105 cpm/assay

conditions

deoxyribonucleoside was used

of the

activity

of pot-

in a standard

of the

from the

tritium.

incorporation

-> 5’ exonuclease

irradiated

at a dose

a dialysis

assays

One unit

inactivation

DNA synthesis

wrong

at 37'C material

each of dATP and dTTP,

phosphorus

measuring

incubation

as a measure

calculated

dose with

enzyme was measured

of concurrent

respect

was labelled

MNU and 0.5 M for

synthesised

by a method

were were

a 137Cs source

Enzyme corresponding

before

newly

materials

The enzymes

using

as a split.

elsewhere'.

of dG into

was

in between.

of the

enzyme was added

incorporation

M for

pH 7.5

from

MNU and DMS was in the presence

(0.1

inactivation

Radioactive

Amersham. of air

with

of DMS were given

phosphate

Details

et a17.

presence

were purchased

Poly(dA-dT).poly(dA--dT)

Centre,

Treatment

buffer

doses

potassium

Ltd.,

of Schachman

the Radiochemical

M NaCl at O°C in the

of 8.4

After

Corporation

triphosphates

soluble

of radioactivity

activity.

RFSULTS AND DISCUSSION Effect

of x-irradiation

activity

of E.coli

dose following

or methylation DNA polymerase

in vitro

to reduce

27 krad,

14mM and 1lOmM respectively

The addition

the

I decreases

V-irradiation

In order

on DNA polymerase

enzyme activity

of MNU or DMS hydrolysis

exponentially

or treatment

with

50% the doses for r-radiation, products

803

I

The specific with

increasing

MNU or DMS (see

required

(D

50

fig.1).

) are

MNU and DMS treatment.

to the enzyme had no

Vol.61,No.2,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

\

.

x

\

x

IO

effect

on

these

dose

activity.

experiments

coinciding The

its

D5'

with quoted

20 [MNU] M&S/I

Inactivation is

apparently

the

change

above

for

30 x lo3

by biphasic

over DMS

is

from obtained

region.

804

40

DMS under with

single-

the to

by

50

the

conditions

change

in

slope

split-dose

extrapolation

of

(see of

the

Methods). single-

BIOCHEMICAL

Vol. 61, No. 2, 1974

Following

a-irradiation

accuracy

of the

enzyme in the

of dG incorporated

polymerase

(see

fig

2).

1 wrong

base

in 200,000

natural

error

of the

doses

detectable The addition on the base

These

incorporation

I-irradiation high

fidelity

is

taken

(O.OOOyb),

as a measure here

of the

a decrease

capable

in the

increasing

to the

Following

template. accuracy

of detecting

to detect

produced

with

products

of the

the

base in 500,000

errors

and increase

enzyme.

is

insufficient

or of MNU the

assays

is also

of a poly(dA-dT).poly(dA-dT)

The assay used

of MNU hydrolysis

fidelity

results

copying

there

enzyme of -ca 1 urong

of radiation in the

BIOPHYSICAL RESEARCH COMMUNICATIONS

and MNU treatment

The amount

high

ANli

of the -ca

reported

(0.0002%)L.

At

by the enzyme become dose

(see

fig

assay had no detectable

DMS treatment

no significant

2). effect wrong

was detected.

show that

the

fidelity

or methylation

with

of DNA polymerase

of DNA polymerase MNU.

I is

It

of E.coli 0-O

proposed"

due to the 3' -> 5'

Total [DMSl Figure 1. Inactivation (b) MNlT and (c) D% of Klenow.

has been

can be lowered

Moles/lx

that

exonuclease

by the activity

IO3

DNA ~~olymerase by (a) a-radiation, DNA polymerase I, X-----X enzyme A

805

Vol.

BIOCHEMICAL

61, No. 2, 1974

Wrong

Base

AND

Incorpwat~on

BIOPHYSICAL

with

l

DNA

y-Irradiated

/

with

is available so,

the

and 3'

-> 5’

enzyme;

to excise

decrease

methylation

the

could

this

any wrong

in fidelity

activity bases

that

exonuclease

activities,

/

/ . ./

DNA polymerases following DNA polymerase I, line indicates the limit

acting

as a "proof"

may be inserted. on J-irradiation

inactivation

the latter

806

Polymerase

A

of the DNA polymerase

be due to a differential

COMMUNICATIONS

I

Enzyme

Wrong base incorporation by E.coli Figure 2. N (a) v-radiation and (b) MNU treatment. The horizontal X-X enzyme A of Klenow. detection in the assays used.

associated

DNA

Polymerase

*Klenow

RESEARCH

being

of the

reader If

of

and

this

is or

polymerase

the most sensitive.

Vol. 61, No. 2, 1974

Since

BIOCHEMICAL

DNA polymerase

interfere

with

I also

the assay

future

work.

cleavage

and it

3' -> 5'

exonuclease.

onlg

Effect

of X-radiation

activity

of E.coli

increasing (see

la

of the Like

and lb).

fidelity

Effect

activity

the

ratio

necessarily

for

the high

fidelity

number

the

an in vitro

activity

first

acti.vity

dose range

also

3" -> 5'

then

If

in exonuclease

report

treatment.

above results

polymerase activity

The results

activity could

doses.

807

here,

to decrease

Z-irradiation to 3'

or MNU

-> 5' exonuclease This

activity

lack

of

activities

is not

does

responsible

which

possesses

were to make a relatively be explained In these

not be detectable

in fidelity

reported

was found

an enzyme molecule

molecules. would

of a decrease

accuracy

The exo-

activity

and exonuclease

exonuclease

no exonuclease

the

the

shows a high

investigated.

of polymerase

but

few error-prone

decrease

is

the

in

at the higher

polymerase following

of the polymerase.

of errors

of relatively extra

inactivation

with

or MNU treatment.

activity

the

and a

and 23 mM respectively

a decrease

polymerase

the cleaved

over

mean that

a good polymerase

exponentially

noticeable

cleaved

of DNA polymerase

constant

selective

not

with

as the polymerase

remaining

detectable

is

particularly

by Z-irradiation

associated

with

decrease

to

The specific

or MNU on the 3' -> 5' exonuclease

activity

treatment

is

I the

can be lowered

at the same rate

This

2) which

of g-radiation

nuclease

large

fig

this

was decided

a DNA polymerase

enzyme A

could

I by proteolytic

a D5C' of 10 krad

or MNU with

DNA polymerase

which

namely

that

enzyme A of Klenow 11,12

so called

enzyme A decreases

Accompanying

it

from DNA polymerase

shows two enzyme activities,

of radiation

intact

obtained

activity

activity

the

or MNU on DNA polymerase

enzyme (see

the

enzyme is

exonuclease

exonuclease

DNA polymerase,

DNA polymerase

doses

fig

cleaved This

a 5' -> 3'

of the 3' -> 5'

use the proteolyticallg for

possesses

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the circumstances in

these

of a DNA polymerase coupled

with

formation

the

the assays.

following

Vol.

61, No. 2, 1974

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

r

presence

of an error-prone

and the possibility the

copying

connection

of carcinogen

of DNA8'1', between

DNA polymerase

are

inaccuracies

in human leukaemic

modified

indicative

that

DNA bases there

in DNA synthesis

cellsb

mis-coding

during

may be indeed

a

and carcinogenesis.

Acknowledgements Miss A. Cogley is thanked for expert technical This work was supported by grants from the Medical assistance. Research Council and the Cancer Research Campaign.

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

Trautner, T.A., Swartz, M.N. and Kornberg, A. (1962) Proc.Nat.Acad. 'ts, 449 - 455. Sci., Chang, L.M.S. (1973) J.Biol.Chem., H, 6983 - 6992. Orgel, L.E. (1963) Proc.Nat.Acad.Sci., 49, 517 - 521. Orgel, L.E. (1973) Nature, a, 441 - 4%. Wheldon, T.E. and Kirk, J. (1973) J.Theoret.Biol., 2, 107 - 111. 70, 245 - 249. Springgate, C.F. and Loeb, L.A. (1973) Proc.Nat.Acad.Sci., Schachman, H.K., Adler, J., Radding, C.M., Lehman, 1-R. and Kornberg, A. (1960) J.Biol.Chem., a, 3243 - 3254. Saffhill, R. (1974) Biochim.Biophys.Acta. $9, 23 - 31. Wang, T.S.F., Sedwick, W.D. and Korn, D. (1974) J.Biol.Chem. 249, 841 - 850. Brutlag, D. and Kornberg, A. (1972) J.Biol.Chem., H, 241 - 2a K. and Patkar, S.A. (1971) Eur.J.Biochem. Klenow, H., Overgaard-Hansen,

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Setlow, P., Burtlag, D. and Kornberg, A. (1972) J.Biol.Chem., 224 - 231. Gerchman, L.L. and Ludlum, D-B. (1973) Biochim.Biophys.Acta.

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&, 08,