A new DNA polymerase activity of Escherichia coli . I. Purification and properties of the activity present in E. coli polAl

BIOCHEMICAL

Vol. 41, No. 6, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A NEW DNA POLYMERASE ACTIVITY I.

PURIFICATION

OF ESCHERICHIA s.

AND PROPERTIES OF THE ACTIVITY

PRESENT IN -E. COLI POLAl Robb E. Moses and Charles

C. Richardson

Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115

Received November 12, 1970 Summary. A new DNA polymerase activity present in cells of Escherichia coli has been purified to homogeneity from E. coli polA1. The new enzyme (polymerase II) differs from thepreviously described polymerase (polymerase I) in chromatographic sensitivity to sulfhydryl-blockinn agents, response properties, to antibody, and template requirements. The predominant (polymerase studied

I)

in extracts

extensively

has promoted activities extracts not

found

(21, with

polA1

deoxynucleotide

of -E. coli

in cells Gefter

reduced

for

responsible

for polA1,

DNA polymerase

(2),

with

sedimenting dient.

a pressure

This

report

a new polymerase distinct describes

activity

from those

the purification

--in vivo.

activity, polymerizing

methods,

purified

(7).

of polA1

the isolation

(polymerase I.

II)

activity

by

on a glycerol

gra-

from E. coli

polA1

which

II

and

and

has properties

The accompanying

of polymerase

(4,5),

Kornberg

a polymerizing

lysate

do

can be detected

in membrane fractions

(6) or toluene

E. -- coli

Although

DNA synthesis

(3),

of polymerase

has been

of polymerase

by conventional

activity,

describes

E. coli

deoxynucleotide

prepared

cell

activity

of a mutant,

DNA synthesis

ether

(8) have partially

levels

additional

in spheroplasts

treated

of wild-type

The identification

markedly

a search

contain

in vivo --

(1).

polymerizing

paper

from wild-type

cells.

MATERIALS AND METHODS E. -- coli

strain

P3478 (polA1)

was a gift

1557

from J.

Cairns

(2).

of

Vol. 41, No. 6, 1970

Strain

DllO

BIOCHEMICAL

(polA1

The standard polymerases

endI-)

has been previously

reaction

I and II

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mixture

contained

(0.3

described

ml)

for

66 mM Tris-HCl

(7).

the assay buffer

(pH 8.0),

6.6 mM MgC12, 1.6 mM dithiothreitol,

0.033

dATP,

, 0.25 mM "activated"

and 3H-dTTP

sperm DNA (9), the reaction activity

(1 x lo7

and enzyme.

After

was terminated

was determined

enzyme activity 10 nmoles of 30 min at

cpm/pmolej

is total

37O.

the

and the

Other

described

amount leading into

materials

for

dCTP, dGTP, salmon

30 min at

37O,

amount of acid-insoluble

as previously

nucleotide

mM each of

incubation

of DNA

to the

(7).

radio-

One unit

incorporation

acid-precipitable

of

of

material

in

and methods have been described

(7).

RESULTS Purification The absence of mits

an accurate

recovery

of Polymerase

polymerase

I in extracts

determination

of polymerase

II

of

during

the

purifications

Table purification

Fraction

specific

of -E. coli

polA1

activities (Table

and the 1).

Units

II

from PolAl Specific Activity units/mg

I. II. III. IV.

Extract DEAE Batch DEAE Column Phosphocellulose Column

(265) 238 179 154

protein (0.03) 0.24 0.35

96

Polymerase II was purified from 20 gms of cells. Table I. (Activity in the extract is not proportional to the amount of protein added to the reaction mixture.)

1558

per-

I

of DNA Polymerase

Total

II

BIOCHEMICAL

Vol. 41, No. 6, 1970

Toluene T-broth paste

(7) --E.

to a density

of

was suspended

1:3

pH 7.4,

for

brought

to

cells

treatment

2 hrs with 20°,

The cell

in the

pellet

10 min.

for

4 months with

no loss

DEAE batch--A

X-100.

above buffer.

a single

(pH 7.4) cell

granular brated with buffer

with

0.02

800 ml of (pH 6.8)

of

a 5-min

was then

-60°

tolueneperiod,

with

diluted

1:3

The suspension

at 6000 lbs/sq. x g, and the

column

in.

was

The extract

supernatant

fluid

(19 cm2 x 27 cm) was

(pH 7.0).

200 ml of

eluate

Fraction

II

was diluted

1:5

from 0.02

had been equili-

gradient

of

M KP04 was pumped through

1559

with

The column was washed

Then a linear

to 0.35

(v/v)

and

to a column of micro-

(18 cm2 x 20 cm) which

above buffer.

were discarded II).

(pH 6.8).

I

by 600 ml of

(Fraction

and adsorbed

M KP04 buffer

the

at

20°

at 0-4O and all

suspension over

was collected

(pH 6.8)

DEAE cellulose

at

and 1 mM EDTA.

to the column and followed

fraction

M KP04 buffer

out

- 5% Triton.

DEAE chromatography--Fraction 0.01

shaker

(w/v)

I).

The first

1600-ml

The

8000 x g at

have been stored

The solution

0.2 M KP04 buffer

(1200 ml) was applied

was then

of 1:4

on a rotatory

stirring

DEAE-cellulose

with

(KP04),

10 min.

15 min at

were carried

with

(Fraction

for

at a dilution

cells

15 min at 30,000

was recovered

for

(200 ml) of the

a pressure

was centrifuged

phosphate

of activity.

was diluted

passed through

and stirred

gently

The cell

The suspension

1 mM 2-mercaptoethanol

0.3 M KP04 buffer

equilibrated

at 4O.

procedures

200 ml of 25% Triton

the

stirring

A portion

cells

and harvested.

in 0.05 M potassium

was re-suspended

subsequent

Extract--

with

(w/v)

by shaking

contained

treated

9 x lo8 cells/ml

The toluene-treated

for

buffers

DllO was grown at 35O in

by centrifugation

same buffer

All

coli

made 1% in toluene,

were collected

4O.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

KP04 the

Vol. 41, No. 6, 1970

column,

BIOCHEMICAL

and 15-ml

fractions

single

were collected.

stituting

the

O-12-0.15

M XPO4 were pooled

peak of polymerase

Phosphocellulose diluted to

1:2

a column

the

(v/v)

(Fraction

0.02

eluting

III

gradient

A single

column

(Fig.

1).

pooled

(170 ml)

buffer

(pH 6.5),

of

the

the

equilibrated

adsorbing

I

I

20

40

400 ml of same buffer

activity

having

and concentrated

to a phosphocellulose

I 80

I 100

0.1 M KP04 from

0.10 were

from the

activity

1:2

with

fractions

eluted

polymerase

by diluting

I 60

and adsorbed

column and ll-ml

peak of polymerase

The fractions

at

(175 ml) was

(pH 6.5)

The column was washed with A linear

con-

III).

M KP04 buffer

M KP04 was pumped over

collected.

activity

(7 cm2 x 30 cm) of phosphocellulose

(pH 6.5).

to 0.40

The fractions

chromatography--Fraction

with

same buffer.

buffer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

in 0.1 M KP04 column

I 120

FRACT/ON

Phosphocellulose

chromatography

of DNA polymerase

II

Z?$Al. (1 cm2 x 5 cm),

and then

eluting

in

8 ml

of

0.35 M KP04 buffer The enzyme

- 1 mM EDTA - 1 mM dithiothreitol.

(PH 6.5) (Fraction

IV)

has been stored

at O°C for

3 months without

loss

of activity. Properties Purity--Fraction during

electrophoresis

merase II,

purified

of Polymerase

IV of polymerase on polyacrylamide from wild-type

cells, 1560

II

II moves as a single gels.

Fractions

are shown in Fig.

band of poly2.

BIOCHEMICAL

Vol. 4 1, No. 6, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 2. Polyacrylamide gel electrophoresis of polymerase II paper). fractions purified from E. coli W3110 (see accompanying Electrophoresis was performan 7.5% gels according to proGels were fixed and stained cedures in the Canalco bulletin. for 8 hours in 0.15% Coomasie Blue in 20% trichloracetic acid, and de-stained in 40% ethanol - 5% acetic acid. A - Fraction I; B - Fraction II; C - Fraction IV.

Table Requirements

for

II

DNA Polymerase

II

Activity

Relative

Relative

Condition

Condition

Activity

Control - DNA, + poly d(~-T) - Dithiothreitol + N-ethylmaleimide 1 $1; 1 ;;:I + 0.3 mM ATP - TTP

Activity

100 <5 75 3 <5 <5 100 7

+ + + +

dGTP, - dCTP 4dXTP, + 4dXDP IdXTP, + 4dXMP 2 @I sodium PPi 0.03 M (NH4)2SO4 10 pg/ml Pronase 20 pg/ml Pancreatic DNase

4 <5 <5 45 40 <2
was assa ed as described in Table II. Polymerase II al$ivity+ Materials and Methods. Mg , Mn , and CaY+ were present at 6.6 mM. N-ethylmaleimide was present at 3 mM. Poly d(A-T) was are expressed relative to present at 0.04 mM. The activities that observed with activated DNA as 100% (0.06 nmoles of 3H-dTMP per reaction mixture). acid-insoluble Reaction nucleoside

requirements--Polymerase

5'-triphosphates

II

and Mg++ (Table 1561

requires II).

all

four

deoxy-

The base compo-

Vol. 41, No. 6, 1970

sition

of the product

Micrococcus tion

BIOCHEMICAL

luteus

of these

obtained

primers.

pH for

KP04 buffer.

with

DNA primers

PPi,

the

is

salmon sperm DNA and identical

The reaction

of N-ethylmaleimide, optimal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of enzyme added and is

inhibited

or pancreatic

reaction

The rate

is

is

to the

by the presence

DNase (Table

8.1 in Tris

buffer,

of reaction

is proportional

linear

time

with

base composi-

(Fig.

II).

The

and 7.5 to the

in amount

3).

a

Figure 3. (a) Time course of polymerase II reaction. Enzyme was present at O.l2).kg/reaction mixture. (b) Proportionality of reaction with polymerase II. Assay conditions are as described in Materials and Methods. Template

requirements--Of

activated

III), greatly

reduced

centrations

a variety

DNA gave the maximal activity

which

(less

allow

than

maximal

of DNAs tested

activity.

Polymerase

5%) with

activity

(Table

poly

with

d(A-T)

polymerase

II

had

at conI.

DISCUSSION Polymerase

II

differs

merase I in several matographic

properties

merases have different inhibited merase

antibody

from the previously

respects:

1) Polymerase

than polymerase primer

(11) .

agents

4) Polymerase

to DNA polymerase

II

(10).

requirements.

by sulfhydryl-blocking I

I

described

I,

(10)

assayed has less 1562

poly-

has different

chro-

2) The two poly3) Polymerase

which

do not

affect

in the presence than

II

20% activity,

is

polyof

BIOCHEMICAL

Vol. 41, No. 6, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Activity

of DNA Polymerase

Template

Activated

Poly

II

T7 DNA

dA

Table III. The standard assay replacing activated DNA in the are expressed relative to that (0.05 nmoles of acid-insoluble More than 95% of the poly d(A-T) the incubation.

whereas polymerase

DNA polymerase merase II

is

II

indicate I.

that

results

from the mutation

It

in polA1.

to polymerase role

accompanying

paper.

E. coli --

in wild-type

found

The possible

mpmoles/reaction

75 30 30 30 12 140 14 65

was used with the various DNAs reaction mixture. All activities obtained with activated DNA 3H-dTMP per reaction mixture). remained acid-insoluble during

In the accompanying

present

Templates

has 95% activity.

to that

identical

Various

100 50
DNA

Our results

using

Activity

DNA

T7 DNA Heat denatured Ml3 DNA Poly d(A-T)

III

of polymerase

II

poly-

in amounts comparable

seems unlikely

purified

does not contain

paper we show that

cells

in polA1, II

polA1

since

that

polymerase

the enzyme appears

from wild-type in vivo --

II

is

cells.

discussed

in the

Acknowledgement. The work described in this paper was sponsored by grants from the National Institutes of Health (AI-06045) and R.E.M. was supported by a the American Cancer Society (P-486). Public Health Service Fellowship (S-F03-GM42,968). C.C.R. is the recipient of a Public Health Service Research Career Program Award (GM-13,634). References. 1. Kornberg, A. Science 163:1410 (1969). 2. DeLucia, P., and Cairns,. Nature 224:1164 3. Smith, D. W., Schaller, H. E., and Bonhoeffer, 226:711 (1970). 1563

(1970). F. J.

Nature

Vol. 41, No. 6, 1970

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

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Knippers, R., and Strbtling, W. Nature 226:713 (1970). Okazaki, R., Sugimoto, K., Okazaki, T., Imae, Y., and Sugino, A. Nature 228:223 (1970). Vosberg, H-P., and Hoffmann-Berling, H. Personal communication, 1970. Moses, R. E., and Richardson, C. C. Proc. Natl. Acad. 67:674 (1970). Sci., U.S. Kornberg, T.,and Gefter, M. L. Biochem. Biophys. Res. comm. 40:1348 (1970). J. Biol. Chem. 237:519 AposhiaK H. V., and Kornberg, A. (1962). Richardson, C. C., Schildkraut, C. L., Aposhian, H. V., and Kornberg, A. J. Biol. Chem. 239:222 (1964). Jovin, T. M., Englund, P. T., and Kornberg, A. J. Biol. Chem. 244:3009 (1969).

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