Studies on the processivity of highly purified calf thymus 8S and 7.3S DNA polymerase α

Vol. 90, No. 3, 1979

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

October 12, 1979

Pages 864-870

STUDIES CALF

ON THE THYMUS

PROCESSIVITY OF HIGHLY PURIFIED 8s and 7.3s DNA POLYMERASE a

K. McKune Biochemistry Centre, Received

July

and A. M. Holmes

Department, Strathclyde 31 Taylor Street, Glasgow

University, The Todd G4 ONR, Scotland

30,1979

SUMMARY Template-challenge experiments indicate no gross difference in processivity of the calf thymus DNA polymerase a A and C enzymes. B 0th Results showing the apparent processive enzymes appear to be distributive. nature of both enzymes on poly (dC). oligo (dG)lo when challenged with poly (dA). oligo (dT)lo are explicable by the failure of both enzymes to bind to the challenging template rather than by the presence of an initiation factor which preferentially binds to certain templates. INTRODUCTION A great deal of interest highly

purified

DNA polymerases,

processively

before

of that chain can continue.

elongation

from

the growin’g

p has been shown to act distributively are altered number

E.coli

chain before

polymerase

a has been claimed distributive

binds

to certain

under

of the enzyme

to the presence templates

during

@I 1979 by Academic Press. Inc. in any form reserved.

of reproduction

(1,3).

under

of an initiation

864

when the conditions more

Rat liver

certain

DNA showing

conditions

which

processive

can be added to

and results

factor

template-challenge

0006-291 X/79/1 90864-07$01.00/O Copyright All rights

KB cell DNA polymerase

monophosphates

to act processively

the

It then has to rebind

I can become

is released

act

that is,

monophosphate

all conditions,

DNA polymerase

the enzyme

nature

chain.

of

I (1.2) and calf thymus

Although

of deoxynucleoside

the growing

been attributed

these enzymes

of a few or only one deoxynucleoside is released

apparent

on whether

E. coli DNA polymerase

DNA polymerase

and an increased

on the mode of action

a and S (2) have been shown to act distributively,

incorporation

of synthesis

lately

especially

or distributively.

DNA polymerase after

has centred

have

preferentially

experiments

(4).

the

Vol. 90, No. 3, 1979

Previously 7.3s,

we

C enzyme,

chain, plus

and

utilisation

8 8,

may

of of

A

have

that

50-70,

in

molecular

in

made.

The

fact

has

led

on these

complexes

weight

material,

be

to alter

able

processes.

DNA

nature

equivalent

to A

in of

these

enzyme

template-challenge

between results

A2 as

and A

MATERIALS

(4)

obtained

enzymes

may

the

using

other

experiments, are

show

us has

be

the

no

polymerase

other

due

to

here.

differences

at which

the

more

that

active

the

50-

activity,

more

of

workers one

the

complexes,

is

speculate

or

in

the

rate

to

by

show

chain

These

A enzyme

one

(I.

polypeptide

differences

of

C enzyme to

reported

in

polymerase

polypeptide

(5).

that

subunit

results

C enzymes

same

in

which

polymerase

Differences

processive

using

the

this

stabilisation

differences

than

molecular

be

and

and/or

link

70,000

of weight,

to

DNA weight

complexes

binding

internucleotide

thymus

molecular

template-initiator

can

calf

consisting

processivity

C enzyme

the

of a 155,000

000

differences

differences

shown

enzymes,

synthetic

reflect

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

consisting

the

material

BlOCHEMlCAl

(2).

using Our

differences

in

A1

gave

enzyme

the on

group

may above

the enzyme

attempts, processivity similar

2’ AND

MET

HODS

All materials were as described (6) except as mentioned Poly (dA), poly (dT), (dT)lO, (dG)lO and (A)10 were obtained from Biochemicals. Poly (dC) was obtained from a (dC)5 initiator using thymus terminal transferase as described (7) and was a gift from Johnston.

below. P.L. calf Dr. I. R.

DNA polymeraseo C enzyme was prepared by the action of urea on enzyme and A 2 enzyme was reconstituted from C enzyme as described DNA polymerase activity using activated DNA was assayed as previously (55. (6), except that the buffer used was 50 mM tris HCl pH 7.8. 1 unit of DNA polymerase activity incorporates 1 nmol of [3H] dTMP into an acid insoluble form in one hour at 37°C. Both enzymes had a specific activity in excess of 50, 000 units/mg on this template.

A

at

30°C

Assays in 0.

using synthetic 13 ml and multiples

template-initiator of this volume.

865

complexes 0.13 ml

were contained

carried 1 mM

out

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dithiothreiol, 62.5 pg bovine serum albumin, 10 mM MgG12, 1 pg of the indicated template-initiator complexes, enzyme protein and the relevant c3H] and [ l*C] deoxyn UC1eoside tri hosphate at 0. 1 mM and 12-15 cpm/pmol for [3H] and 3-5 cpm/pmol for [ 18 C]. These assays were carried out at either pH 6.4 in 20 mM sodium-potassium phosphate, or at pH 7.8 in 50 mM tris HCl. The template-initiator complexes were prepared and assays processed as described (6). Template -challenge experiments were carried out in multiples of 0. 13 ml and samples were withdrawn at intervals as described in the relevant figure legends. The complexes used were:poly (dA). oligo poly (dT). oligo (A)lo (T:A = 1:l) and poly (dc). oligo (dT) 1o (A:T = 20:1), (dG)lO (C:G = 5:l). The base ratios used are at or close to the optimum for both A and C enzymes. RESULTS

AND

DISCUSSION

Preliminary

template

ascertain

the effects

utilisation

of others

shown poly

in Table (dC)

using

of several

Although

1.

(dA).

(dC). oligo

Further

experiments

support

the incorporation

not used incorporation. utilised,

(dT)

reduced

Neither

poly

(dA) calf

When were

time

carried

(dC).

poly

thymus

added.

calf poly

A and

oligo (dA).

does

(dT) 10 -s on dGMP

DNA

was being

p01y (dT).

A and C enzymes

oligo

on this

had any effect

(dT)lo

C enzymes.

(dG)lo oligo

thymus

(dC),

both of dTMP,

and had no effect

of A and C enzymes addition

inhibited

results The

(dG)lO,

oligo

(dC). poly

activated

of both

is negligible

is

(A)lo

template.

on the utilisation

DNA.

out at pH 6.4,

Similar

of dFMP when

An example

by both

that

out to on the

the incorporation

pH poly

showed

oligo

(dA).

courses

(Fig.

was

at which

of dGMP,

nor

of dGMP

inhibited

carried

complexes

as template-initiator,

the activity

immediately

(dC)

(dG)lo

at pH 7.8,

incubation 1).

oligo

of poly

or poly

of activated

the incorporation

(dT)lo

were

A and C enzymes.

the incorporation

Also addition

purified

at pH 7.8,

to support

experiments

of the template-initiator

by highly

and poly

poly

competition

were

reciprocal

of poly

on poly (dC).

the incorporation obtained

oligo

866

oligo

(dG),,

of dTMP

in the absence

experiment

(dA).

to the by both

of dGTP

at pH 6.4,allowing

(dT)lo

enzymes

or when synthesis

poly to

Vol. 90, No. 3, 1979

TABLE

Templates

BIOCHEMICAL

PRELIMINARY

1

present

TEMPLATE

~01~

and

dG)

oligo( poly(dC)

dT) . oligo(

pm01

by

by

by

1o

10 dG)

poly

A

C enzyme

[ 14C]

dGMP

incorporated by

A

134.5

C enzyme

0

0

0

0

2

2.5

0

0

0

0

25.5

31

4

2

29

36.5

0

0

1o

~ol~(dA).oligo(dT)l~ and

EXPERIMENT

incorporated

(dC)

poly(dA)

COMPETITION

180

. oligo(

RESEARCH COMMUNICATIONS

prn~l[~H]dTMP

~ol~(dA).oligo(dT)lO poly(dC)

AND BIOPHYSICAL

(dC)

Assays were On activated C incorporated

for DNA

10 min. at pH 6.4 as described in MATERIALS A incorporated 132 pm01 [3H] dTMP and 93 332 pm01 [3H] dTMP and 222 pmol [14C] dGMP

Figure 1 Template poly (dC), oligo (dG) incubation contained intervals and processed

challenge experiment showing 10 on ongoing poly (dA) replication 0.39 ml and 50 ~1 samples were as described (6). The mix

[ 14 C] dGTP . The arrow (a) 2 units of A enzyme o-o

shows the (b) 5 units

v-v

poly (dA). oligo added after 30 oligo 3 ccg POlY (U). (dG)lO added after 30 3 pg poly (dC). oligo

v-v

water 3 pg

0-O

3 pg water

PT)lo

added after 30 min. poly (dC) oligo (dG) 1 o added added after 30 min.

Incorporation

of

[ 14C]

dGMP

the

effect at pH withdrawn

contained of the incubation

time of addition of C enzyme per

(dT)lO added min. (dT) 1 o added min. (dG)lo added

was

867

AND METHODS. pm01 [14C] dGMP in the same time.

of addition 6.4. Each at 10 min.

[3H] challenging

at

0 min.,

at

0 min.

at

0 min.,

sterile

at

0 min.

, 2 kg

poly

(dA).

and

not

shown.

negligible

sterile ,

2 pg

is

glass poly

(dC). glass

dTTP

and

of

and template.

distilled oligo distilled oligo

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 2 Template challenge experiment showing the effect of addition of poly (dA). oligo (dT)10 on ongoing poly (dC) replication at pH 7.8. Each incubation contained 0.52 ml and 50 ~1 samples were withdrawn at 10 min. intervals and processed as described (6). The mix contained [3H] dGTP and [14 C] dTTP. The arrow shows the time of addition of the challenging template (a) 1.45 units of A enzyme (b) 2.6 units of C enzyme per incubation. o-o

4 ug poly (dC). oligb (dC)lO water added after 40 min. 4 pg poly (dC) . oligo (dG) 1 o added after 40 min. (dTjlo oligo (dT)lO 4 t% POlY (dA). water added after 40 min. 4 pg poly (dA) . oligo (dT) 1 o added after 40 min. WC),,

0-a V-V v-v

Incorporation

begin

on poly

showed

that,

poly

(dC).

(Fig.

1).

addition

(dC).

oligo

although oligo

When

dTh4P

(dG)lo

(dG) 1O inhibited

(dA).

enzyme

oligo

1 and 2 and the preliminary

suggest

that (dT)lo

preliminary synthesis in agreement

at 0 min.,

added

at 0 min.

, 2

negligible

challenging was

the incorporation

was

those

DNA

is distributive.

of other

workers

(dC).

and

shown.

is not

poly

negligible,

oligo

by both

of

enzymes

out at pH 7. 8, the

(Fig.

2).

of dGMP The

data

in

experiments

at pH 6.4 (dC). would

(dT)lO,

the presence

on incorporation

These (4).

oligo

(dA).

competition

experiments

distilled

, 2 pg poly

carried

incorporated template

glass

of dTMP

was

distilled

pg poly (cL4). oligo

with

of dGMP

glass

sterile

at pH 7. 8 on poly

competition

on activated with

added

sterile

by A and C is distributive

and is processive template

at 0 min.

(dT) 1 o had no effect

figures

oligo

added

experiment

and no dTMP

synthesis

at 0 min.,

was

then

incorporation

the reciprocal

of poly

by either

of [ 14C]

added

on poly

oligo

(dG)lo.

would (dA). The

also

tend

to suggest

results

are

substantially

However,

when

synthesis

that

on

Vol. 90, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

b

Figure 3 Template challenge experiment showing the effect of addition of on ongoing poly (dC) replication at pH 7. 8. Each poly (dT). oligo (A)10 incubation contained 0.52 ml and 50 ~1 samples were withdrawn at 10 min. intervals and processed as described (6). The mix contained [3H] dGTP and unlabelled dATP. The arrow shows the time of addition of the challenging template (a) 1.5 units of A enzyme (b) 1.65 units of C enzyme per incubation.

o-o

4 pg poly (dC). oligo (dG)10 water added after 40 min. 4 kg poly (dC). oligo (dG)lo added after 40 min. (A)10 4 pg poly (dT). oligo (A)lo water added after 40 min. 4‘pg poly (dT). oligo (A)10 added after 40 min. (Wlo

O-0 V-V v-v

poly

(dC).

oligo

incorporation

(*)lo

that

synthesis

template

challenge

(dT).

oligo

When

indicate

synthesis by

both

C to

activity

A and of

template-initiator

calf

the be

at

thymus

to

in DNA

complexes

2 pg poly

at 0 min.,

sterile

added

at 0 min.,

2 pg poly

the

[

14

[

14

oligo

a distributive polymerase are

not

on

due

869

distilled

(dC).

oligo

(dT).

(Fig.

3).

oligo

(dG)lo.

the

C can

(dT). (dG),,

oligo the

manner.

Thus

a A and

C enzymes

to any

oligo

gross

oligo This Further

indicate

that

addition

of poly

template

A nor poly

(dT).

poly

present

preliminary

on

(dC).

(dC).

C ] dAMP

7. 8 neither

poly

poly

C ]dATP

Also

procede

of

inhibited on

distilled

glass

addition

immediately

processive

pH

glass

added

shown).

of

acting

at 0 min.,

incorporate

allowed

addition

added

with

not that

was

challenged

not

to

(data

(A)lo

experiments

is

begin

sterile

by

was

experiments

C enzymes

at 0 min.,

challenged

of dGMP

indicates

A and

was

(dG)10

added

both

binding

bind

to poly (A)

1.

(dA).

and

was

results

again

showed

the

difference

in

differences

on various in

Vol. 90, No. 3, 1979

proce

s sivity,

evidence

BIOCHEMICAL

or lack

that

templates.

there The

of it,

but as A enzyme

(5),

may

A dangerous that when

that

this

whereas

challenged

template,

but fail

initiator

to demonstrate

of a DNA

polymerase

molecule

described

by Chang

may

contain

certain

factor(s)

involved

active

than

C on all

the above

templates

for

to bind

Also,

binds

made

type

and a [14C ] of [3H]

being

in

on a challenged

processive

may be distributive

out using

on the on the

template.

the processive

deoxynucleoside

to certain

of experiment

on incorporation

be carried

no

a factor.

to the challenging

unequivocably

we have

preferentially

in this

polymerase

the enzyme

should

enzymes.

such

has no effect

is due to the DNA

template,

nature

which

is usually

template

challenged

experiments

factor

preference

assumption

a challenging

template

two

is more

be no template

RESEARCH COMMUNICATIONS

these

000 material

initiation, there

between

is any initiation

50-70,

AND BIOPHYSICAL

Further

or distributive a [3H]

or [ 14C]

triphosphate

as

(2).

ACKNOWLEDGEMENT This

work

was

supported

by a grant

from

the Medical

Research

Council.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

Uyemara, D., Bambara, R.A. and Lehman, I. R. (1975) J. Biol. Chem. 250, 8577-8584. Chang, L. M. S. (1975) J. Mol. Biol. 93, 219-235. Bambara, R.A., Uyemara, D. and Choi, T. (1978). J. Biol. Chem. 253, 413-423. Fichot,-E, Pascal, M., Mechali, M. and De Recondo, A.-M. (1979) Biochem et Biophys Acta 561, 29-41. McKune, K. and Holmes, A. M. (1979) Nucleic Acid Res. (In Press). Holmes, A.M., Hesslawood, I.P. and Johnston, I.R. (1974) Eur. .I. Biochem. -43, 487-499. Bollum, F. J. (1966) Procedures in Nucleic Acid Research (Cantoni, G. L. and Davis, D.R. eds) pp 577-583, Harper and Row, New York.

870