A novel single strand endonuclease specific for ØX174 DNA

Vol. 67, No. 4, 1975

BIOCHEMICAL

A

NOVEL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SINGLE

STRAND

SPECIFIC

FOR

Shalini

Singh

Received

October

0x174

and

Department Molecular Los

ENDONUCLEASE DNA

Dan

of Biology

S.

Ray

Biology

and Institute

UCLA California

Angeles,

90024

28,X975 SUMMARY

A highly specific endonuclease activity, presumably involving one or both of the products of the 0X174 gene A, has been isolated from 0X174infected E. co1 i by DNA-ccl lulose chromatography. The enzyme is not present ix uninfected cells and binds extremely tightly to DNA-cellulose. It extensively degrades 0X174 viral DNA but does not degrade the circular or linear forms of single stranded viral DNA of either M13, an unrelated fi lamentous phage, or G4, a 0X-type phage. Replication DNA

of

requires

functions and

the

the

function

provided is

of

by

necessary

viral

the

for

and

a

0X174

amber

and

shown

to

cleaving

the

viral

strand

ability

was

also

phage

(1,2).

The

accumulation

of

to

mutant have

a

to

0X174

gene

nick

have

gene

A

the

RF

at

0X174

in

addition

is

-cis

wild-type

The

single

gene

both A

acquires wild-type

protein

In

site.

on

a

has

been

activity

the

activity

to acting

RF

endonucleolytic a

0X174

containing

with

supercoils,

endonucleolytic

of

protein

infected (4).

(RF) A,

molecules

specific

0X174

A

Only cells

gene

gene RFII

in

highly

of

specifically

found

in

form

gene,

(3).

discontinuity

isolated

its

single

discontinuity

strand-specific

0X174

a

replicative

host

the

strand-specific

this

double-stranded

addition

purified

0X174

(5), to enzyme

single-stranded

DNA. The of

35,000

an

internal

translational

and

A

GO-65,000

two

(6).

initiation level

produces

within (7).

Both

protein

The

smaller

the

A

products

gene

products protein at bind

having appears

either tightly

a

molecular to

result

transcriptional to

DNA-cellulose

weights from or and

Vol. 67, No. 4, 1975

only

elute

BIOCHEMICAL

with

liminary

buffers

containing

characterization

present

in

the

2.0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of M NaCl

2.0

the

wash

M NaCl.

0X174 of

single

We report strand

DNA-cellulose

MATERIALS

AND

here

a pre-

endonuclease

activity

columns.

METHODS

Bacterial and phage strains: r. co1 i ~560 (F+, as the host for all preparations of 0X174-infected used were: 0X174 mutant; -am 3, a lysis-defective type G4.

thv

, end cells. wild-type

A,

pal A) was used Bacteriophages Ml3; and wild-

Endonuclease Assay: The enzyme activity was assayed by incubating enzyme fractions with circular sinqle stranded viral DNA, at 37% in 0.2 M TrisHCI pH 7.5, 10 mM MgC12, and - 0.1 M NaCl for 20 minutes. At the end of the reaction the loss of biological activity (infectivity) of 0X174 DNA was determined by spheroplast assay (8). The percentage of DNA molecules inactivated in treated DNA samples was calculated on the basis of the reduction in plaque formation as compared to that from untreated DNA samples. me Purification: E. coli ~560 was grown to a density of 2 x 108/ml in H-broth, infected wTthm74 am 3 at a multiplicity of infection of The cells were then centrifuged 4-5 and allowed to grow for 2 hours at 37°C. at 10,000x for 5 min at room temperature and resuspended at a concentration of 0 x 101 3 cells/ml in 10% sucroseand 50 mM Tris-HCl pH 7.5 at room temperature. Soluble extracts were prepared by a modification of the method of Wickner The ccl Is were first subjected to rapid --et al (9). freezing followed by a slow thawing at 0-4’C. The cells were then incubated at 0°C for 30 minutes with IOOug lysozyme per ml in the presence of 0.5 M NaCl and 5mM spermidine, followed by 2 minutes incubation at 37'c+ The lysate was centrifuged at 40,000 rpm in a Beckman 65 rotor for 15 minutes at 4OC. The supernatant was then dialysed against buffer containing 0.2 M Tris-HCl pH 7.5, I mM EDTA, 10% glycerol and 0.1 M NaCl. The dialysed cell extract was precipitated by adding ammonium sulfate to 40% saturation. The precipitate was resuspended in the same buffer containing 0.4 M NaCl, and dialysed overnight against this buffer to remove ammonium sulfate. The dialysed protein was applied to a 4.5 Cm by 9 mm diameter column of denatured calf thymus DNA-cellulose pre-equilibrated with a buffer of 0.2 M Tris-HCl pH 7.5, I mM EDTA, 10% glycerol and 0.4 The column was then washed with 2 bed volumes of buffer containing M NaCl. 0.4 M NaCl, followed by a second wash with 2 bed volumes of buffer containing 1.0 M NaCl. Most DNA-binding proteins eluted at this step. Finally, a third wash of 2 bed volumes with buffer containing 2 M NaCl eluted the single strand endonuclease. This fraction could be stored in the presence of 10% glycerol without loss of biological activity. Alkaline samples

Velocity in alkaline

Sedimentation: sucrose

gradients

Sedimentation of was performed

enzyme-treated as previously

DNA described

RESULTS The

enzymatic

columns

loaded

in

1.

lated

Fig.

more

activities with

The than

ability 4-fold

of

extracts

from to

in

the

2 M NaCl

infected

inactivate eluates

eluates

and

infectious from

0X174-infected

1430

of

DNA-cellulose

uninfected 0X174

cells

viral cells.

are DNA

is

compared

stimu-

(10).

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

QUANTITY

FIGURE I: 2

0X174 eluates

M NaCl

or uninfected infectivity

To of

0x174

or

G4

specificity

the

0x174

was

slow

linear

sedimenting

Thus,

the

Z(a)

degraded

by

the

At

strands

and

reaction

a

G4

single

form

we

that

the (o---o) for

present

Yet

both

can

be

viral

of since

treated 0X174

linear

the in

the seen

as

a

strands

in

samples

(Fig.

viral

DNA

Ml3

Ml3

and

G4

(d) In

both

top

of same

and

the

cases the

G4

DNA

on the

2(b)

the

untreated and

does

single

sucrose

reaction

shoulder both

treated

demonstrate

the

probably

fate Ml3

the

and

at

the

either

sedimented

2(b)

remained

and

included

strands.

strands

the

have and

single

that

and

activity

Fig.

0X174

circular

(c))

(d)). not

strands

involve

were

not

enzyme. of

intermediate shorter amount

each

to

degradation

degradation

digest.

in

for

the

exonuclease

The

increasing

and

extensive

non-specific

manner

conditions.

of

enzymatic

this

molecules

side

(Fig.

nor

the

gradients.

enzyme

these

2O-30%

samples

DNA

degraded

under

0.15, and 0.20 ml of from 1 liter of infected DNA was then assayed

in

sucrose

Ml3

degraded

of

treated

this

Neither

contained

specificity

alkaline

DNA

The

viral

of

gradient. were

the

stranded

through

0,0.05, columns protein-treated

(ml )

spheropiasts.

strands

single

with

cells.

both

single

the

was treated DNA-cellulose

(a--o) to bacterial

examine

mixture

a

DNA from

OF ELUATE

strands of

enzyme

0X174

DNA

levels

of are in

seen the

seen

in

Fig.

degradation as reaction

1431

2

is

(Fig. products

of the

apparently 3) digestion.

weight-average

a

limit

unit-length

linear With molecular

Vol. 67, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

-

W

Fraction

IO

20

30 40 Froctlon

number

IO number

20

30

40

Specificity of single-strand endonuclease activity. 2: Treated and untreated DNA samples were sedimented through alkaline sucrose gradients for 6 hours at 4°C and 56,000 rpm in a ~~60 rotor. (a) Untreated Five-drop fractions were collected from the bottom of the tube. 3*P-labeled 0X single strand (o-o) and 3H-labeled Ml3 single strand (b) DNA of (a) in equal concentrations (1010 molecules/0.2ml) (o-e) DNA; luate from DNA cellulose; treated for two hours with 8 pg protein from 2 M NaCl G4 single (c) Untreated 3H-labeled 0X single strand (m--o) and 3s P-labelpg (d) DNA of (c) in equal concentrations (10 molecules/ strand (o-o) DNA; 0.2 ml) treated for 2 hours with 8 pig protein from 2 M NaCl eluate from CNA-ccl lulose. FIGURE

3:

Intermediates in the endonucleolytic degradation of 0X174 strands. 3*P-labeled 0X single strand (o---o) and 3H-labeled Ml3 single strand DNA molecules/O.2 ml) treated (-) DNA in equal concentration (lOlo and 8 (d) ug protein from 2 M NaCl for 20 minutes with 1 (a), 2 (b), 4 (c) eiuate from DNA-cellulose.

FIGURE single

1432

BIOCHEMICAL

Vol. 67, No. 4, 1975

weight

of

the

reaction

products

rema

i ned

was

AND BIOPHYSICAL

decreased.

RESEARCH COMMUNICATIONS

Again

Ml3

DNA

contained

0X174

single-stranded

in

the

same

undegraded. DISCUSSION

A

novel

purified

endonuclease

from

0X174-infected

degradation

of

for

0X174

that

is

or

most

single G4,

in

might

initially

the

to

of

that the

DNA

and

as enzyme RF

this

the

to

molecule

be

in

sequenceby

one

DNA or

the 0X of from

the

a

0X174

least

in

processive

so

recognized

results

discriminate

properties

bacteriophage,

to

frequency at

specificity

between

Alternatively,

of

DNA

and

more

and 3

only

the

enzyme

Fig. a

degradation

degrade do

not

single

unique

products

between

endonuclease

extremely

possible

necessary

only

gene

strand

A of

a

single

A.

and site.

to --in

is

is

in

mechanisms

cells,

the vivo

of

involved

The

of (5)

extensive

1433

of

from

or

both

action

of

gene

A

the

specific

at the

a

single

RF.

site Similarly,

0X174-infected

degradation

suggest

one

in RF

specificity

columns

action the

replication

Knippers

its

DNA-cellulose

double-stranded for

Henry

to

due

Yet

function the

prerequisite by

is

gene

0X174-infected

binding

activity

The viral

in tight

0x174

the

purified at

is

DNA

sequence

basis

,

degradation

a

not

sequences

However

enzymatic of

the a

the

size.

nucleotide

us

appears

random

characterization

its

controlled. of

a

of

filamentous

high

similar

rare

of

observed

nicking

enzyme

high

discriminate

unrelated

since

on

extremely

degrade

been

enzyme.

products

highly

an

has

endonucleolytic

combination

to

extremely

found

allow

presence

the

with

which

should

the

0X174

Ml3,

processively. in

this

endonuclease

of

of

the

specificity

of

detailed

and

The

occur

recognize

A more

action

an

DNA

extent

and

indeed

The

species

strand

progress

is

can

those

be

DNA

a model

site.

It

the

enzyme

endonuclease

likely

single

the

sufficient

and

have

other

support

nicks

have

Both

by

bacteriophage?

would

times

the

yet

restriction-type

therefore

of

DNA

How

strands

would

a.

unexpected.

a 0X-type

enzyme

for

viral

puzzling. and

specific

(3.4)

are

for

i.

0X174

DNA

extensively 0X174

specific

cells of

0X174

viral

is

Vol. 67, No. 4, 1975

DNA of

observed gene

free

here

A on

viral

even

strands

before The

reflect

are

single

not

natural

of

substrate

by

the

enzyme

should

requirements

of

the

enzyme

and

in

an

of

both

of

infected

of

the

cell

0X174

encoated

detected a supercoiled

regulate

allow the

or

products since

infection

with

capsid

here

might

(11). proteins

DNA molecule.

degradation

the

the

be

conceivably

extensive

one

stage

enzyme

portion

of

of

to

the

purification

MW products

any

progeny

for

RF might

preventing

at

appear the

of

found

present

single-stranded

structure

activity

normally

strands of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

an

not

completion

a transient

protein

might

a substrate

0x174

Nascent

BIOCHEMICAL

role

of us

to

of

both

RF the

activity

the

viral

probe

I.

The

the

strand.

35,000

gene

A

Further

both and

be

duplex

of

further

the

possibly

the the

substrate 60-65,000

A gene.

ACKNOWLEDGEMENTS This of

Health

research (Al

was

supported

by

a grant

from

the

National

Institutes

01752).

REFERENCES

(1965) Virol. 2, 303-321. Tessman, E.S. Tessman, E.S. (1966) J. Mol. Biol. 12, 218-236. Francke, B. and Ray, D.S. (1971) J. Mol. Biol. 61, 565-586. Francke, B. and Ray, D.S. (1972) Proc. Natl. Acad. Sci. 2, 475-479. Henry, T.J. and Knippers, R. (1974) Proc. Natl. Acad. Sci. 71, 1549-lS53 Linney, E.A., Hayashi, M.N. and Hayashi, M. (1972) Viral. 0, 381-387. Linney, E.A. and Hayashi, M. (1973) Nature New Biol. 3, 6-8. Sinsheimer, R.L. (1968) Methods in Enzymology 12, 846-858. Edited by Grossman, L. and Moldave, K. (Academic Press, New York). Wickner, W., R. Schekman. K. Geider and A. Kornberg. (1973) Proc. Nat. Acad. Sci . , Vol. 70, 1764-1767. (1971) Virol. 44, 168-187. Francke, B. and Ray, D.S. (1968) Prog. Nucleic Acid Res. Mol. Biol. 8, 115. Sinshimer, R.L.

32: 4. 5. 6. L:

9. IO. II.

1434