The release of oligonucleotides by theEscherichia coli B restriction endonuclease

Vol. 68, No. 2, 1976

BIOCHEMICAL

The Release Escherichia

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Oligonucleotides

coli

Margaret

by the

B Restriction

Kimball

Endonuclease

and Stuart

Linn

Department of Biochemistry University of California Berkeley, California 94720

November 14,1975

Received

SUMMARY: The Escherichia coli B (Eco B) restriction endonuclease releases approximately 75 nucleotides as acid-soluble oligonucleotides for each single-strand endonucleolytic scission that it catalyzes. This reaction, like the endonucleolytic cleavage, requires ATP, Mg*, S-adenosylmethionine, and unmodified DNA containing appropriate specificity sites. Like the endonuclease reaction, the release of oligonucleotides terminates after roughly 5 minutes. The acid-soluble oligonucleotides have an average chain length of roughly 7, and an apparently random base composition.

INTRODUCTION: The restriction

endonuclease

of E. co7.i B is

a complex

Mg*,

and DNA containing

specific

ATP, S-adenosylmethionine, have not

been modified

enzyme appears

to make only

to a DNA-dependent for

up to several

in its

by the

hours

symmetry

(6)

and it

randomly

of this

is

fragments

smaller

On studying reaction--the presents enzyme,

that

than

evidence

that

break All

roughly

of small, these

pairs

acid-soluble

It

also

gives

fragments.

Copyright All righis

G 1976 by Academic Press, Inc. of reproduction in any form reserved.

but

is known

585

itself

phosphate

restriction

nucleases

the sequence

has no sequence,

concerning

the location

upon,

or to form duplex

to act (4,9).

enzyme we have observed

oligonucleotides

not by a contaminant.

most

The

converts

the DNA at the specificity

reluctant

1500 base of this

unlike

sequence,

that

the enzyme is

then

which

(l-3).

ATP to ADP and inorganic

a unique

(4,7,8).

sequences

methylase

scission,

enzyme is

does not

the products formation

This

recognizes

but more or less cleavage

hydrolyzes

(4,5).

It

modification

one endonucleolytic

ATPase which

specificity:

obvious

complementary

enzyme requiring

DNA fragments. are

formed

another This

enzymic report

by the restriction

some characteristics

of the

Vol. 68, No. 2, 1976

MATERIALS

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS:

Restriction enzyme was the sucrose gradient fraction, unless otherwise indicated, and was prepared as described previously (1). One unit of enzyme causes 0.6 pmol of single-stranded DNA scissions (1). The preparations of 3H- or 32P-labelled DNA from E. coli, 3H-labelled DNA from phage x and [y-32P]ATP were described (1,lO). Techniques used to characterize the oligonucleotides were as described by Goldmark and Linn (10). Restriction reactions (70 MgC12, 0.5 mM DTT, 1.4 mM ATP S-adenosylmethionine, 1.5 nmol for 15 min at 37'. ATPase and acid-soluble (1) - DNA rendered

~1) contained 70 mM Tris-HCl (pH 8.2), 7 mM ([y-32P]ATP was used for ATPase assays), 14 pM DNA, and restriction enzyme. Incubations were endonuclease assays were as described previously was determined by adding 50 1.11 5 mg/ml bovine

TABLE I The release

Experiment I

II

III

of acid-soluble

Assay

DNA during

the restriction

pm01 Acid-soluble nucleotide formed

conditions

complete

Nucleotides released per DNA strand scission

- enzyme

co.2

78 --

- MgC12

<0.2

--

- ATP

<0.2

--

- S-adenosylmethionine

<0.2

--

+ i-0

16.3

reaction

DNA

+ h-0 DNA (denatured)

<25

84 --

+ X'B

DNA

<25

--

+ 0.11

unit

PA Frn.

4

4.9

75

+ 0.14

unit

PA Frn.

7

5.9

69

+ 0.18

unit

SG enzyme

8.6

78

209

3H-labelled E. cozi K DNA (10,600 cpm/nmol) and 0.35 unit of enzyme used in Experiment I. but scaled up to 700 1.11, were used Reactions as in Materials and Methods, in Experiment II. Phage 1 DNA grown on a non-modifying strain of E. coZi (A.0 DNA) or grown on E. cozi B (X-B DNA) had 1360 cpm/nmol and 1210 cpm/nmol, respectively. E. coli K DNA (10,200 cpm/nmol) and enzyme as shown were used in Experiment III. "PA" enzyme is that eluted from polyacrylamide gels. Fractions 4 and 7 were those containing the two separate peaks of enzyme activity separated by electrophoresis on these gels, and were obtained from the particular gel pictured and assayed in Figs. 1 and 2 of reference 1. "SG" enzyme is sucrose gradient enzyme. were

586

BIOCHEMICAL

Vol. 68, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

serum albumin and 0.25 ml 7% trichloracetic acid to a chilled incubating for at least 10 min at 0'; then centrifuging for and counting an aliquot of supernatant (10).

reaction, 5 min at 25,000

x g

RESULTS AND DISCUSSION: Characteristics

of the

restriction

reaction:

per

single

strand

requirements

as the

endonuclease:

In addition,

the acid-soluble

the B modification; modifying

methylation

the reaction

is

upon which

the

not

time

course

reaction

(1).

reaction

characteristics

are

is

This Mg*,

material not

formed

by growth observed

material

75 nucleotides

scission.

the restriction

of acid-soluble is

it

exposed

roughly

endonuclease,

material

When DNA is

essentially

of endonucleolytic

In this

way the release

as acid-soluble

has the same cofactor

observed

only

from DNA which B host

complete

with

(Table

DNA (Table

after noted

of acid-soluble

(2). 5-10 for

(Table

DNA not

has received

does not act

scissions

B (Eeo B)

and S-adenosyomethionine

heat-denatured

endonuclease is

is

in an E. coli

with

released

reaction ATP,

to the E. cozi

I).

containing

the appropriate In addition,

I and Fig. Finally min

2 below)

the release (Fig.

1).

This

the restriction

material

has the same

as the endonuclease.

--~---a

IO

20

I 30

Time (mln)

Figure 1. Time course for release of acid-soluble nucleotide. The reaction was as described in Material and Methods, but scaled up to 402 ul. 3H-labelled E. coZi K DNA (10,600 cpm/nmol) and 2.8 unit of enzyme were used. Fifty ul aliquots were removed at times shown and acid-soluble radioactivity was determined.

587

I).

Vol. 68, No. 2, 1976

BIOCHEMICAL

Association of the activity

with the Eco B restriction

of purification

of the

restriction

gradient,

yields

a preparation

which

independent

preparations

same level

during

restriction activity the

removed

the sedimentation,

releasing

acid-soluble

is

associated

in this

step

When the Eeo polyacrylamide the gel of activity

with

relative

by its

as homogeneous

enzyme

was assayed

for

Sedlmentoton

subjected

of activity

Frachon

assayed

(six)

showed

ATPase

of acid-soluble

with (Fig.

the (The

2).

behind

which

on denatured

is DNA.)

to electrophoresis

1).

the In

trailed

activity

acts

totally

activity.

DNA that

can be detected

2 of reference

the release

All

co-migrated

also

a sucrose

(1).

contaminating that

step

75% pure

from duplex

a separate,

(Fig.

through

to endonuclease

DNA-dependent

enzyme is

two peaks

were

activity

of the purification,

gels,

about

that

this

enzyme: The final

sedimentation

is

material

B restriction

-

that

of nucleotides

enzyme as measured

ATPase

enzyme is

of such material

of release

addition,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

upon

and eluted

from

Each of these

peaks

nucleotide,

and

Number

Figure 2. Preparative sucrose gradient sedimentation of restriction enzyme. DEAE-cellulose enzyme fraction was further purified by preparative scale sedimentation through a sucrose density gradient as described by Eskin and Linn (1). Gradient fractions were assayed as described in Materials and Methods with E. coli K DNA (3H-labelled, 10,600 cpm/nmol for DNase assays), or, for ATPase assays, [Y-~'P]ATP (108 cpm/nmol). One unit of ATPase hydrolyzes 100 nmoles of ATP in 15 min (l), whereas one unit of DNase is defined for this figure as that amount rendering 100 pmol DNAnucleotide acid-soluble.

588

BIOCHEMICAL

Vol. 68, No. 2, 1976

found

to be identical

(Table

I).

with

sedimentation this

pH 3.5 shows

both peaks of restriction

endonuclease,

(Fig. is

part

that

all

counts

and nucleosides

DNA as with

soluble

counts

Chromatography

of acid-soluble

of dinucleotides

When the acid-soluble

bacterial

alkaline

by virtue

reason,

larger

the average

more than Analysis

than

75 nucleotides

significantly

at

the cathode). with

Therefore,

on paper

In

32P-

the acid-

By this

criterion

is

7.

length

It

product

may be released DNA upon sucrose

were

3'P-labelled

phosphate

with

the average

chain

should

be noted

(11).

For

may be larger,

during

a strand

scission.

gradients

gives

no evidence

material

that

we have excluded

in length

of small

more non-sedimentable

from

products,

15 nucleotides

in the regions

No mononucleotides

to inorganic

thus

in c-propanol-

appearing

was isolated

of acid-soluble

chain

of restricted

material

and larger.

product

roughly

confirm

nucleotide

is noted

DNA.

material

phosphatase.

of our selection

material

material

be converted

of the acid-soluble

with

toward

in the radioactivity

32P could

I)

the

form.

to octanucleotides

DNA, 14.5% of the

with

electrophoresis

the anode

[3H]thymidine-labelled

resulted

(Table

Paper

migrate

of the acid-soluble

NHI+OH-1120 (6:3:1),

observed.

toward would

are in nucleotide

coupled

oligo-

enzyme.

material:

migrate

fraction to release

requirements

of the restriction

the same amount

labelled

length

2) and reaction

of the acid-soluble

(Bases

addition,

sucrose-gradient

of the ability

data

Characterization

to the

RESEARCH COMMUNICATIONS

migration

activity

material.

regard

The electrophoretic

nucleotides

that

in this

AND BIOPHYSICAL

than we observe

this and indeed,

for

as acid-soluble,

however. 32P-labelled with

pancreatic

acid-soluble

four

nucleotides;

of the substrate

was also

DNase and venom diesterase.

of the mononucleotides the

product

showed instead

no evidence

Separation for

the base content

DNA.

589

digested

to mononucleotides and quantitation

a predominance appeared

of any one of

to reflect

that

Vol. 68, No. 2, 1976

BIOCHEMICAL

ConeZusions:

results

The above

made by the Eco B restriction

AND 8lOPHYSlCAL

demonstrate enzyme

is

roughly

75 nucleotides

as oligonucleotides

random

base composition.

The time

shows

that

phase

of the restriction

for

the release

as long

with

the product

situation

(9) have

suggested it

specificity

sites

hypothesize

that

traverse DNA.

the

released.

able

to act

restriction

in

upon

cleaving

If

this

were

upon another

molecule,

enzyme added

to a reaction

since

endonucleolytically

before

Whatever

of oligonucleotides,

for

the source

in developing

models

for

the

the case,

it

DNA below

"fall

off"

complex

represent

I,

which

Horiuchi,

even minimal

before

the

size

they site,

cleaving we have

that

the observed

still

be

each

cleaves

the ATPase

state

Eco B restriction

by the

though

ultimately

presence

et al.

length

enzyme would

into their

the

event

they

hand,

that

"fallen"

mixture

continues

whether

endonuclease

we have observed

dissociates

the

is

DNase

which

the specificity

the oligonucleotides

off"

1)

enzyme molecules.

degradation,

but

phase

to a minimal

recognize

of the molecule,

7 and

DNA endonucleolytically

restricted

With

of

the early,

or whether

On the other

intact.

"falling

(Fig.

fragmentation

E. coZi

with

to further

still

formation

of restriction

observed

the enzyme molecules

length

Perhaps

are

are

length

question

scission,

once DNA is

resistant

chain

the ATPase

of a single

communication).

that is

during

by a number

40 nucleotides

release

during

A noteworthy

as part

events

personal

Eco B enzyme,

not

(4,5).

has been

roughly

(Wm. Studier,

occurs

the endonucleolytic

of multiple

The former releases

hours

are released

simultaneous

of their

scission

by the

of average

course

reaction,

each single-stranded

accompanied

of oligonucleotides

as several

oligonucleotides

that

RESEARCH COMMUNICATIONS

DNA (1).

must be accounted reaction.

ACKNOWLEDGMENT: This

work

was supported

by U.S.

Public

Health

Service

Grant

GM19020.

REFERENCES: 1. 2.

Eskin, B., Linns, S., 1300-1306.

and Linn, and Arber,

S. (1972) J. Biol. Chem., 247, 6183-6191. W. (1968) Proc. Nat. Acad. Sci. USA, 59,

590

Vol. 68, No. 2, 1976

3. 4. 5. 6. 7. 8. 9. 10. 11.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Roulland-Dussoix, D., and Boyer, H.W. (1969) Biochim. Biophys. Acta, 195, 219-229. J.A., Eskin, B., and Lackey, D. (1974) Linns, S., Lautenberger, Fed. Proc., 33, 1128-1134. Eskin, B., and Linn, S. (1972) J. Biol. Chem., 247, 6192-6196. Dugaiczyk, A., Kimball, M., Linn, S., and Goodman, H.M. (1974) Biochem. Biophys.Res. Comm., 61, 1133-1140. Adler, S.P., and Nathans, D. (1973) Biochim. Biophys.ActA, 229, 177-188. Horiuchi, K., and Zinder, N.D. (1972) Proc. Nat. Acad. Sci. USA, 69, 3220-3224. Horiuchi, K., Vovis, G.F., and Zinder, N.D. (1974) J. Biol. Chem., 249, 543-552. Goldmark, P.J., and Linn, S. (1972) J. Biol. Chem., 247, 1849-1860. Cleaver, J.E., and Boyer, H.W. (1972) Biochim. Biophys. Acta, 262, 116-124.

591