A DNA-dependent ATPase from E.coli infected with bacteriophage T4

Vol. 41,No.

BIOCHEMICAL

1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A DN>1-DEPCMDPNT ATPase

lu'ancy Cebr:-cm-i, Cancer

Research

Margaret

Laboratory,

August

T; Se'nm2 and I;. Ebisuzaki University

Lcndon,

Received

FRO14 E.CC*iI

Ontario,

of Western

Ontario

Canada

24, 1970

Sumnary: A DNA-dependent ATPase has been purified from --E.coli infected with ---.-^ The enzyme has a 10~ molecular weight and is not detectbacteriophage T4. able in uninfected E,coli, In the presence of DNA, ATP is clcavcd to ADP and inorganic phosphate, Calf thymus DNA, --E .coli DNA and heat-denatured !'L, and T7 bacteriophage DNA's stimulate the ATTPase. However, native T4 and Ti DNA's do not activate the enzyme. In addition to ATP breakdown, the enzyme catalyzes dATP and, to a lesser extent, CTP degradation to their respective diphosphates, No exe- or endonucleolytic activity has been detected. The role of DNA i.n the reaction is currently under investigation, In this of

communication

the properties

temperature cells

:itre

tant,

Solid and the

ethanol, which

incubated

found

for

by 0.05

by chromatography

by the

by rupturing

purification

in extracts

and some

of T4 bacterio-

hj- 0.14 addition

T4 am EGO5

at a multiplicity

of 4-5.

cells

and the

to 55% saturation

resuspended after M N&l,

in 0,OlM dialysis,

was then

M potassium plasma

albumin

115

supernatant

was added tris

The

at

pressure was re-

to the

superna-

pH 7.5 + O.OlM mercaptocelluln;:e.

chromatographed

phosphetc

and stored in a French

on DF&

on hydroxylapstite.

of bovine

withbacteriophage

centrifuged, the

sulfate,

ammonium sulfate precipitate,

mutant)

25 minutes,

streptomycin

was chromatographed,

hydroxylapatite

was infected ligase

prepared

with

was eluted

followed

l-)

-sensitive

extract,

was treated

covered.

ized

SH22 (endonuclease

A cell

cell,

ATPase

on the

and Methods

(an amber,

-20°C.

studies

E.coli. ---

F ccl?. LO.'.-

infected

our

of a DNA-dependent

phage-infected Material

we report

The enzyme,

on S,sphadex

The ATPase was eluted buffer,

pH 6.83,

(final

concentration,

C-50, from

and was stabil1 ma/ml).

VoL41,No.

Details

1,197O

of

BIOCHEMICAL

the enzyme purification

The standard

assay

will

(0.15

mix

MgC12s 3.3 mM; dithiothreitol, (specific

activity

enzyme.

One unit

29.6 is

concentration fraction

was used

in all

leneimine W were

cut

out

Chicago

gas flow Y32P-ATP

Results

p9 7.6,

mM; 14C-ATP,

mM; and 0.05

- 3.0 units

of

0.5 mlJmoles

linearly

units.

67 mM;

3.5 /JM

to produce

varies

to 3.0

by thin

with

with

enzyme

The hydroxylapatite

layer

1 M LiCl

chromatography

(la).

on polyethy-

ADP spots

of 14 C-ADP was determined

and the amount

visualized

using

by

a Nuclear

counter.

cellulose

resolved

by thin

layer

solvent

system

1 (lb).

using

chromatography

on poly-

and Discussion

DEAE-cellulose

ADP-forming

chromatography

activity

was absent

weight

near

15,000

behaviour

on Sephadex

G-100.

presence

of DNA and Mg*

activity

nucleoside

participate similar

in

under

a forty-fold

phate

formed.

other

nucleoside

equimolar DNA is not

quantities labelled

can be assigned Complete

standard

between

reduction

occurs

triphosphates

for

on the

The optimum

ability

ATP and dATP are

cleaved

Inorganic (Table

or S- 14 C-ATP.

116

I.

A

its

their

pH

to to

When CTP is used of nucleoside

of diphosphates

tested.

from

1).

7.3 and 8.5.

in the amount

to the ADP produced by Y32P-ATP

in Table

conditions.

production

--E.coli;

of the reaction

tested

II).

assay

M NaCl on

(Figure

to the enzyme

data

were

(Table

E0 coli

dependence

shown by the lies

at 0.05

of bacteriophage-infected

of uninfected

triphosphates

No detectable

was eluted

of extracts

buffer

the reaction

extents

strate

is

in tris-HCl

Other

activity

in extracts

molecular

for

0.013

corrmunication.

experiments.

developed

A DNA-dependent

this

tris-HCl,

of enzyme necessary

of 0.05

and 32 Pi were

ethyleneimine

contained

DNA, 0.073

was analyzed

cellulose

in a subsequent

ADP formation

the range

ADP formation

ml)

mc/mmole);

at 37°C.

over

appear

11 mM; ATP,

the amount

of ADP in 30 minutes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

were

phosphate III).

as sub-

diphosfound

is

for

the

released

in

No AMP is

formed.

Vol. 41,No.

1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(a) 1

DNA+ -

100

200

,-J#,-

*--a

DNA*

-

300

Eluate volume,

ml

FizO 1. DUE-cellulose chrotilatography of extracts of (a) uninfected, .-. and (b) Tf,atiGO5-infected Ek2.2. The amtilonium suifaie preparaticr!s --E.coli were chromatographed on 1.5 x 10 cm. columns of DEAE-cellulose, with 1 1, 3-chambered concave g.radients of 0 - 0.8 $1 NaCl, in 0.01 M tris-HCl, O.OlPl mercaptoethanol (ph 7.6). 6 ml fractions were collected every 10 minutes, and assayed for AD.? formation in the presence and absence of caif thymus DNA.

Different

DNA substrates

as shown

reaction,

in Table

active

than

native

E.coli -__

sRNA

was substituted

No exonuclease produced in alkaline

no

change sucrose

vary

In every

IV.

DNA; native

in their

case,

T7 DNA and native for

DNA no enzyme

or endonuclease in

ability

activity

the sedimentation

to stimulate heat-denatured T& DNA were

activity

gradient.

117

DNA was more inactive.

When

was noted.

was detected,

of 14C-labelled

the

Enzyme treatment heat-denatured

T7 DNA

BIOCHEMICAL

Vol. 41, No. 1, 1970

AND BlOf’HYSlCAL

mpoles

1. DNA

Expt. Expt.

2. Mg*

RESEARCH COMMUNlCATlONS

ADP produced

-DNA 1 (0.04 2 to.04

+DNA 1.00 0.66

-m*

+I@++

(0.04

0.57

+Mg++ + EDTA (0.04

Table I. Dependence of ATPase activity Standard assay on DNA and Mg*. conditions were used with denatured calf thymus DNA. A 2.5 fold excess of EDTA was used in the reaction mixture.

voles

nucleoside

diphosphate

Expt. 1 0.76 0.76 < 0.08 co.5 0.02 (0.14 to.01 (0.08

ATP dATP GTP dG'IP CTP dCTP UTP TTP

produced

Expt. 2 0.39 0.33 CO.08 (0.5 0.01 to.14 (0.01 (0.08

Table II. Participation of Different Nucleoside Triphosphates in the Reaction. The appropriate nucleoside triphosphates were substituted for ATP in the standard assay using denatured calf thymus DNA. Many of the labelled nucleoside triphosphates used here were contaminated with the corresponding diphosphates and the < values are equivalent to the level of this contamination.

In order precipitated soluble the

with

for

was found.

of ADP produced

V, Expt.

exonuclease

C13CHCOOH after

radioactivity

amount

(Table

to screen

1).

This

activity

undergoing It

is

was in excess fact

together

118

the

14C-labelled

the

assay

noteworthy

that

of the with

the

amoullt lack

procedure; in

these

DNA was no acidexperiments

of DNA-P present of nuclease

activity

Vol. 41,No.

BIOCHEMICAL

1, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ADP, mpmoles Expt. Expt.

1 2

Pi,

mqoles 0.37 0.18

0.36 0.16

Table III. Products of the Reaction. Pi was measured by the release of 32Pi from Y-32P-ATP and ADP by the production of 14C-ADP from 14C-ATP, as described in Materials and Methods.

mlJmoles ADP produced Type of DNA Calf

Native

Heat denatured

thymus Expt. Expt.

1 2

0.32 0.18

0.63 0.33

E.coli --

Expt. Expt.

1 2

0.33 0.26

0.62 0.70

T7

Expt. Expt.

1 2

T4

Expt. Expt.

1 2

to.04 (0.04

0.35 0.82

0.02 0.05

0.65 0.97

Table IV. Effect of the Type of DNA on the Reaction. Equivalent concentrations of the different types of DNA were used under standard assay conditions. DNA samples were heat-denatured by heating at 100°C for 5 minutes followed by rapid cooling to 0°C.

DNA mpmoles Expt. Expt.

1 2

P

0.48 0.11

ADP mi*moles 0064 0.28

Table V. Relationship between concentration of DNA present and ADP produced. In Rxpt. 1 14C-labelled, heat-denatured T7 DNA was used. The DNA concentration was determinrd by optical density, using an extinction coefficient of 6750 (9).

119

Vol.41,No.

BIOCHEMICAL

1,197O

suggests

that

of DM

the enzyme

present

has an unusual

has a marked

ability

of DNA to stimulate

anionic

character.

reaction

is

lated

T4-induced

because

are

the type

reacti.on,

the

be due to its

well

known

enzyme may have

of the

enzyme do not

poly-

in the enzyme

to attack

a unique

and polynucleotide

(3)

by Battin

degrades

the

role

DNA-dependent

in genetic

ATPase;

activity

however,

glusosy-

in the

and in

bacteriophage

mutants

normal

of enzyme 44,

45,

its

47,

52,

(7)

of ATP for degrades

active

enzyme differs fcr

for

55,

native

the presence

these 58,

The ---Micrococcus every

an excess

and Barbour is

& Clark

in

DNA.

Thus

far

of the ATPase

59, 60,

62,

(8)

on T4 DNA as is ours

mutants

of ATP

exonucle-

from

include

known

such as

The ATP-dependent

recombination

investigated

46,

Oishi

preference

activity;

(4).

enzyme

activity. (6),

of the

in Dh'A metabolism, ligase

our

their

to those

3 molecules

Similarly,

& Wright

a role

may have

levels

involved

has no endonuclease

ase described

43,

(5)

bond broken.

contrast,

nucleast

correspond

kinase

phosphodiester

tide

few enzymes the

endonuclease

42,

simply

T4 DNA functions

enzymes

weikticus

41,

of the

cannot

cofactor-requiring

polynuclectide

which

reaction

Since

of action.

of T4 DNA.

The properties

but 'in

mechanism

on the extent

denatured

Therefore

T4 DNA (2).

metabolism

effect the

That

remarkable

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in genes

its

our

exo-

all show 30,

13, 39,

V and X and poljmucleo-

kinase. The ability

large

amounts

nuclease

T4 phage-induced,

of ATP and dATP in the

activity,

physiological DNA in

of the

role

the reaction

suggests of the are

that enzyme

currently

the

DNA-dependent presence

of DNA, without

enzyme has an unusual

in DNA metabolism under

ATPase

to degrade detectable

function.

and the participation

The of

investigation.

Acknowledgements: The authors wish to thank Mrs. --_ the bacteriophage mutants for the presence of the for discussions and help with the manuscript, and Greenberg, J. Hosoda, W. Harm, J. Wiberg and V.L. bacterial and phage strains.

120

Cecilia Senior for testing enzyme, Dr. J.A. KcC;rter R.S. Edgar, J. Eigucr, G.R. Chan for providing tbe

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 41, No. 1, 1970

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