DNA synthesis in cell-free extracts of a DNA polymerase-defective mutant

BIOCHEMICAL

Vol. 40, No. 6, 1970

DNA

SYNTHESIS

IN

CELL-FREE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EXTRACTS

Thomas

OF

Kornberg

and

Department

of Columbia New York,

Received

August

A DNA

POLYMERASE-DEFECTI

Malcolm

L.

Biological University New York

VE MUTANT

Gefter

Sciences 10027

4, 1970

Summary. A DNA-synthesizing activity has been isolated from an E. mmutant defective in DNA polvmerase. Like DNA polvmerase. the svstem requires the presence of all fbur’deoxynucleoside triphosphates, magnesium ion and a native DNA template for maximal activity. The activity can be distinguished from r. coli DNA polymerase on the basis of its sensitivity to high ionic strength and to p-chloromercuribenzoate. The activity is insensitive to antiserum directed against E. col i DNA polymerase. Our results do not exclude the possibility that the activity isolated is composed of, in part, an a I tered form of DNA pol ymerase. tNTRODLJCTION The

isolation

of

investigations --In

into

vitro

the

synthesis in

describe

the

isolation

Pol

Ai

the

extracts

polymerase-defective

nature

using

activity

from

a DNA

of

the

DNA

agar-embedded

of

Pol

ccl

A;

and

mutant

cells Is

col

W3l IO thy-

and

were

were ethanol, ded inchz).

was of

two

Debris

--in

membrane-associated

vitro

of

a soluble

DNA

synthesizing

of and

obtained

IO5

to

several strain.

(2,3).

We wi I I activity

the unbroken

E-,

in

of

O.OlM; above

buffer cells

g. Dr.

from

col

DNA

P.

Research

A;,

A4

(temperature

g.

coli

its

DNA

3

parent sensitive

polymerase,

polymerase

Setlow.

(4),

and

H-thymidine

anti-

tri-

and

was

adjusted

+

or

Pol

A-,

frozen

pH8.2,

0.0005M. lysed

removed

1348

Pol

tris-acetate

E.D.T.A., and

were

Purified

Pol

to

a

mu mole.

of

and

strr, Pol

thy+,

Bio

extracts a solution

lac-,

Gross.

from

per

METHODS

rha-,

Schwarz

cpm

cell-free

Mg(OACj2,

J.

from

x

twice

AND

fragment

were

of

volumes

Dr.

tryptic

I.0

washed 0.005M;

in

this

reported

W3l 10 met

from

purchased

preparation

thawed,

of

been

W31 IO thy-,

142:

obtained

activity For

112:

antiserum (TTP)

specific

capacity

recently

properties

JG

active

polymerase

phosphate

JG

At

enzymatically

DNA

an

led

mutant.

strains

Pol

polymerase) an

i

and

has

have

MATER I ALS E.

synthetic

(I)

in by

The

a pressure centrifugation

0.02M; cells

cells 2-mercaptowere

cell

(9,000 at

(4-59)

12,000

resuspenIbs/ XG.

Vol. 40, No. 6,197O

The

supernatant

30

min.

solution

and

100,000

the

XG For

cerol

for

90

nol, 0.5M.

of

pH

dGTP,

dATP,

40mu

moles

The

reaction

I

of

DV mole

3

DNAase

into

by

H-TTP,

15

35,000

XG

centrifugation

for

at

the

as

an

insoluble

served

the

as

bucket

of

liquid

the

form controls

for

5 or

30

TCA

and

the

The

dCTP, Thymus min.

acid

fi

were

lters

at

were

30°C.

driec

One

the

incorporation

Incubation

mixtures

generally

DNA,

insoluble

counter.

5 min.

and

Spinco

contained

Calf

catalyzes

in

was

SW40

(5)

papers.

which

a

Z-mercaptoetha-

(0.3ml)

scintillation

amount

in

0.3umoles;

were

fiber

giy-

concentration

mixtures

cold

IO-30%

rpm.

“activated”

of

glass a

KCI

Z-mercaptoethanol,

addition

in

and

the

40,000

reaction

moles;

on glycerol

0.005M

each at

layered

dissolving

except

Incubations

GF/C

acid

by

hrs.

IOmu

defined

(75ug/ml)

on

2umoles;

determined

TTP

layered

enzyme.

Whatmann

is

same

for

was

MgS04,

the

activity,

and

on

4’C

MgCl2, each;

radioactivity activity

was at

terminated

0.05M;

were

enzymatic

moles

was

at to

SIOO

prepared

KCI,

solution

of

the

were

gradients

nucleotide,

enzymatic

served

O.OlM;

was

collected

taining

pH7.4,

ZO~moles;

IOmp of

material the

gradients

protein

7.4,

centrifugal-ion

subjected

activity,

salt

salt

measurement

tris-HCI

by

solution

enzymatic

centrifugation

For

and

High

and

clarified

further

RESEARCH COMMUNICATIONS

(SIOO).

tris-HCI

0.5ml

rotor

min.

Low

0.002M.

was

supernatant

of

gradients. of

AND BIOPHYSICAL

resulting

purification

solution

BIOCHEMICAL

~10%

of

unit

of of con-

the

ob-

activity. RESULTS Cell-free

subjected

to

activity bulk

was protein

activity nium

sulfate

dient

buffer for

I and

hr.

(SIOO)

was

centrifugation

as

present

as

(Fig. peak

dient fer

extract

and

(85%

saturation).

(one

tenth

at

subjected

The

4’C. to

the

original dialyzed

centrifugation

low-salt

glycerol

Materials

and

sedimenting

protein

to

the

was

volume) solution as

dissolved and

dialyzed

was

then

described

1349

faster

by

than

two

the

thirds

of

precipitation in

the

layered

+he

with high

against

above.

and Enzymatic

Methods.

middle

concentrated

pellet

gradients

slightly

corresponding

The the

in

peak

Fractions

pooled

on

described

a discrete

IA).

were

layered

salt

the on

Enzymatic

gra-

same

a high

ammo,

salt

activity

bufgra-

Vol.40,No.6,

BIOCHEMICALAND BIOPHYSICALRESEARCH COMMUNICATIONS

1970

5

IO

15

5

I5

IO

FRACTION NUMBER Figure gradients. tation determined

was

I.

Sedimentation of protein in low salt (A) and high salt (6) glycerol Conditions for sedimentations are described in the text. Sedimenfrom right to left. The enzymatic activity of each fraction was Incubations were for 30 min. under standard assay conditions.

is

present

Fractions

as

a discrete

containing

ten-fold

by

preparation

sedimenting

enzymatic

precipitation was

0.002M

peak

dial

dithiothreito

with Y zed

for

.

Th is

slower

activity

were

ammonium

sulfate

2 hrs.

than

pooled

against

the

and

as

the

was

used

protein

protein

described

O.OlM

(Fig.

The

enzyme pH7.4,

tris-HCI

al I experiments

for

IB).

concentrated

above.

o If

a solution

preparation

bulk

to

be

des-

I ly

20-

cribed. The 30.+

specific

Thirty

preparation at

+

O°C

for

Specific fold higher o” c.

ac

ivity

to

forty

percent

and

had

a specific

one

activity than

month

20

without

(units

per of

the activity

significant

measured immediately but decayed to this

mg

protein)

total

of

activity of

after value

1350

was The

600. loss

the

of

SIOO

was

genera

recovered

preparation

activity.

obtaining the after storage

in cou

I ncorporat

final

the Id

be ion

stored of

TTP

SIOO was often several for several hours at

Vol. 40, No. 6,197O

8-

‘t:

u-3 -

co u3

0 v

M co

--

-

I I

N r-

Vol.40,No.6,

1970

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

Table I. Requirements and properties of enzymatic activity from Pol A; cells, Pelt cells, and purified E. col DNA polymerase. The complete system IS as described in Materials and Methods and was the same for all three preparations. Incubations were for 30 min. at 300C. Values in parentheses represent final concentrations in the reaction mixtures. *The activity from Pelt cells was purified using the same gradient procedures as described for Pol AT. Pal+ activity sedimented 20% faster than the activity from Pol AT in the low salt gradient and essentially the same as Pol AT activity in the high salt gradient.

was

linear

for

30

Incorporation to

what

I min.

addition, dependency The

not

The

Pal+

magnesi

ion, from

KCI

and

The

activity

than

the

are

and

E.

Pol

*

from activity

from of

has

shown

the

requ

i rements.

and

the

Pal+

cells

activity that

from it

is

to

the

units).

decline.

In

incorporation

of

dependence native

mixture

ceased

enzyme,

and

presence

leads

enzyme

the

DNA

as

of

in ATP.

This

to

are

In

in

to

are

the

of

at

least

ten-fold

or

DNA

polymerase.++ cells

approximately

IOO-fold

Pol

A;

anti-DNA

I.

As

anti-DNA

shown,

triphosphate, the

presence

polymerase sensitive

is

preparation

activity, the

acti-

mixture

Table

by

less

a thiol

of

deoxynucleoside

more

with

loss

reaction

in

stimulated

presence

A;

the

the

all

and

a similar

the

contrast

of

template

complete

summarized to

presence

p-chloromercuriben-

preparation,

respect

Pol

on

or

antiserum

polymerase

is

a source

concentration)

this

by

began all

on

ion,

with

DNA

cells

as

polymerase

similar

then

(Z-20

preparation.

polymerase

DNA

concentration

and

dependence

reaction

are

A;

used

final

of

enzyme

preparation,

a complete

completely Pol

final was

(0.2M

coli

cells

inhibited

Titration antiserum

properties

DNA

hrs.

final

anti-DNA

The

and

2-3

magnesium

in

of

preparations

activity

the

KCI

(0.3mM)

cells,

urn

SIOO

shows

of

presence

al I three

++The merase Higher below p.c.m.b.

presence

effect.

from

in

activity

(p.c.m.b.)

without

the

triphosphates,

The

in

a three-fold

evident

4 deoxynucleoside

vity.

when

showed

range for

with

incubation

enzymatic

reagent.

increase

observed

system

was

a ten-fold

to

was

of

the

zoate

over

continued

contrast after

min.

of antiserum.

to

p.c.m.b.

polymerase sensitive

than

is

DNA

fifty percent reduction in activity observed with the Pal+ and DNA polyc.m.b. concentration of 3 x 10v5M. preparations is also observed at a concentrations of p.c.m.b. >3 x IO -9. M do not reduce the level of activity 50%. The observed inhibition is believed to be due to the inhibition by of exonuclease ffT present in these preparations.

1352

BIOCHEMICAL

Vol. 40, No. 6,197O

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

25 0

ANTISERUM ( pl 1 Figure 2. Titration of enzymatic activities with anti-DNA polymerase antiserum. Part A represents titration of activity in the SIOO extracts of -8. Part B represents Pol AT (49 units) + and Pal+ (290 units) titration of partially purified activity from Pol AT (3.7 units) -#-8, tryptic fragment of purified E. coli DNA polymerase (3.3 units) r. coli DNA polymerase (4.0 units) + and a mixture of Pol Al activity (3.7 units) and tryptic fragment (4.0 units) .+ . The dotted line represents the theoretical amount of antiserum-resistant activity present in Enzyme and antiserum were incubated with the components of the the mixture. in the absence of deoxynucleoside standard assay mixture at 30°C for 5 min. triphosphates and DNA. DNA and deoxynucleoside triphosphates were then added and incubations were for 5 min. (Part A) and 30 min. (Part 6) at 3ooc. Reactions done in the absence of antiserum were also incubated at 30°C prior to the addition of the other components.

polymerase Pol

or

A;

(Fig.

the

extract

does

tryptic

not

reaction;

presence the

SIOO

extracts

Calf

Thymus

effect.

protect

DNA

DNA

of

DNA

polymerase

Pal+ (33

and mu

final also

i strain

and

polymerase.

The

against

observed

in

the

A;

cells

of

JG

presence

inhibition

of

The

of no SIOO

this

strain

Pol not

by

have

and

in

the

render

same

it

of

antiserum

purified

1353

antiserum

observed

addition

of

in

sodium

sensitive

to

is

desoxycholate

from

cell

activity

The is

to to

as

activity. preparation

extract?,

sensitive

insensitive

specific

without

antiserum.

activity the

the

“activated”

reaction

relatively

polymerization partially

be

with

renders is

the

antiserum

synthesizing

it the

The the

affect also

activity

A4 mutation

antiserum-sensitive and

A;

a DNA

25OC

not

2A). to

the

Pol at

do can

nucleotide)

purified

142.

SIOO

(Fig

does

partially

the sensitivity

concentration)

(MMS)

show

of

treatment

Extracts

extracts

Pol

moles

methylmethanesulfonate 37OC.

components antiserum

addition,

(0.4%

col

the

differential

We have E.

of

of

In

(DOC)

at

fragment

28). The

of

active

not

M.M.S.

Pol

A; activity

cold-

BIOCHEMICAL

Vol. 40, No. 6, 1970

sensitive.

its

insensitive

to

properties

are

antiserum

and

AND BIOPHYSICAL

identical is

to

sensitive

the to

RESEARCH COMMUNICATIONS

activity

0.2M

KC1

from

Pol

to

p.c.m.b.

and

A;

ccl

Is;

it

(0.3mM).

DISCUSSION We have mutants

partially

defective

in

DNA polymerase and

is

in

in

extracts in

polymerase

results

reported

by

not

due

of

DNA

polymerase.

Knippers

to

of

an

A;

cells

activity

to

apparent

ionic

is

not

a

of

of

chain

two

g.

ti

distinguished

from

and

Since

mutant

result

be strength

antiserum.

non-amber

from

can

high

polymerase

polymerase,

the

DNA

to

p.c.m.b.

activity

is

polymerase

(Pal

termination

within

A4), the

not

DNA

expect

expect

result

associated have

enzyme.

been The

determinant

sulfate

or

p.c.m.b.

in

extracts

If

0.5M would

KCI

in

can

of masked but

is to

both

our

Pol

or

that

hands,

and

be

to

A;

ccl

a

fragment

reported

by

the

activity

Is

in

exists,

explained

in

resolve. cells.

then “free”

it

is the in

1354

protein

in both

Pol

have

not

dissociable

presence terms

to

of of

polymerase

dissociation

unl

ikely would

detected

However,

one as

such

antimight

a membranemutants

active

in 0.2M

membrane

anti-

polymerase

be

or

We have

but

DNA

polymerase

part,

enzyme

would

I) is

DNA

DNA

exists,

a mutant

that of

to

wild-type

a complex

I)

another

unrelated

inactive

of:

mutants

naturally

that

result

We believe

to

of

inactive be

a

with

difficult

having such

be

independent

a complex

as

have

to

complexed

polymerase

such

selected

is

of (5)

activity

since

activity

extracts

Kornberg

enzyme.

yet 3)are

properties

genie

clear

two

2)and

DNA

complex.

this

polymerase

and

if

in

by

described

that

activity

this

not

polymerase-like

present

described

DNA-synthesizing

Hypotheses

serum-resistant

be

of

have

properties

genitally.

to

DNA

bound. we

a new

would

identical

is

membrane

3)

residual

relationship

a mutant

or

(I) as

(3)

that

2)

one

Cairns

Stratling not

the

polymerase”

activity

component,

that

The

is

The

by

sensitive

anti-DNA

and

“DNA

and

cells

could

is

indicate

DeLucia

is

have

The

activity

gene.

These

since

Pol

a DNA-synthesizing

polymerase.

it

to

activity

Al

DNA

that

insensitive

present the

purified

“complex”

that DOC,

KCI.

the

4M ammonium The

of

anti-

the

inactivation complex

is

BIOCHEMICAL

Vol. 40, No. 6,197O

and

subsequent

suggest be

that

denaturat such

el iminated.

purification decide

Acknowledqement. Cancer Society.

of

a complex Although

of absolutely

ion

this

exists we

between

“mutant” but

favor

activity

This

the

AND BIOPHYSICAL

the

from

work

was

the

possibility

existence

2)and

supported

We have

pol ymerase. of of

wild-type

hypotheses

RESEARCH COMMUNICATIONS

a new

cells

is

grant

# E-561

no

its

existence

DNA

synthesizing

essential

in

evidence

to

cannot enzyme, order

to

3).

by

of

the

American

References. (1) (2) (3) (4) (5) (6)

De Lucia, P. and Cairns, J. Nature. 224:1164 (1969). Smith, D.W., Schaller, H.E., and Bonhoner, F.J. Nature. -226:711 Knippers, R. and Stratling, W. Nature. 226:713 (1970). Brutlag, D., Atkinson, M.R., Setlow, P. and Kornberg, A. Biochem. Res. Comm. 37:722 (1969). Aposhian, H.rand Kornberg, A. J. Biol. Chem. (1962). -237:519 Kornberg, A. Science. l63:1410 (1969).

1355

(1970). Biophys.