An endodeoxyribonuclease induced in E. coli by infection with λpbio transducing phages

Vol. 41, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AN ENDODEOXYRIBONUCLEASE INDUCED IN E. a- COLI BY INFECTION WITH WBIO Andrew Department

Received

August

TRANSDUCING PHAGES

Becker

of Medicine, University of Toronto, the Ontario Cancer Institute, Toronto, Canada.

and

21, 1970

Summary. Infection of g. coli by @& transducing phages leads to the production of a DNA endonucleolytic activity. The activity has not been detected in uninfected cells, nor in cells infected with ?, phages that do not carry the bio-containing, bacterial DNA segments. The endonuclease has been purified extensively. The purified enzyme catalyzes the production of double-strand breaks in DNA molecules. Genetic

recombination

of DNA molecules the control viral

activities

bearing

phages

mutants

in which

proceeding

endonuclease

rather

than

with

communication Materials

Signer

describes

).

infected

that

the genes

of E.

of the an

the endonucleo-

A, and with

?, particles

of the phage

chromosome.

transducing

attachment (3).

and

Unexpectedly,

a

the Xpbio's

with

recombination.

properties

h

site

infected

controlling

and certain

region

under

we compared

of biotin

markers

are

to demonstrate

at the prophage

in extracts

the purification

phage

- a set

by bacterial

carry

region,

segment

beginning

are replaced

non-essential

with

and rejoining

processes

In attempts

of this

the lw's

was encountered J. phages

so-called

recombination-subserving

A genes,

by the breakage

X, recombinational

(2)

the control

of X used were

to the right,

occurs

on the

of E. -coli

contiguous

potent

This

of this

enzyme.

and Methods Strain

Berling

of DNA under

in this

linked

by E.R.

of extracts

deletions

The deletion

of genes

(reviewed

breakage

viruses

In the case of phage

of a series

chromosome

enzymatic lytic

(1).

in bacterial

(4).

1100 is Other

Most of the hpbio

the endonuclease g. coli

phages

were

I-deficient

K strains the

gift

were

obtained

of Dr.E.R.

63

isolate from Signer.

of Durwald Drs.

and Hoffman-

C. Fuerst

Phage

Lint red 4-3

and B. Rolfe. was the

Vol.41,No.

gift

of Dr.

M. Pearson,

from

BIOCHEMICAL

M. Yarmolinsky

(5).

yields its

infection

of h for

prophage

growth

0.02

M Tris

then without

in flasks

were

ation,

then

Protein

C-labeled

cooled

polyallomer

in x DNA.

manner,

mixtures

and protein.

counting.

and 0.01

weight

conditions one unit

Activities

The assay products where

from less

of activity

than is

after

were

in the cold

during

in 0.2 M

to 2-3 x lo8 cells

to 0.02

M.

Phage were

10 min at room temperature another

20 min at 37'.

cells

harvested

pH 7.0

In the early

The

by centrifug-

I ( 8 ).

were

layered

0.5 ml water 14

the percent

the distribution

amount

64

calculated

of substrate in the

25% of the substrate the

solutions

catalyzing

for

30 min

gradients

contained

in

1.0 M

DNA was sedimented by collecting

and 5 ml Aquasol

C and 3H were

( 10 mM ),

A DNA was then

on 5 to 20% sucrose

fractionated

at

0.14

( New England

and plotted displaced

with

ml

Nucthe aid

to smaller

gradient

of unreacted

molecules

are displaced

the breakage

of

&WA

was at 30'

).

of

20 mnmoles

of purification,

Tritium-labeled

sucrose

were

the production

in 0.1 ml,

Incubation

The sucrose

gradients

for

( 50 mM ), MgC12

stages

EDTA ( 50 mM ).

with

for

assayed contained,

M BDTA ( neutral

of

scores

out

14C or 3H-thymidine,

was added

for

), Tris

endonuclease

vials

with

in L.B.

infected

Incubation

180 min at 5' and the

of a computer.

the

of a Beckman SW 56 rotor.

scintillation

sulfate

cells

The mixtures

pH 8.0,

DNA was carried

bacteria

vigorously

500 cpm/mnmole

to inhibit

of kc1 -B57s-7 by lytic

prepared

(7) and stored

from these

'as marker.

rpm for

Under

E'uerst,

at -20'.

by adding

M Tris

into

agitated

was terminated

tubes

0.05

molecular

Drs.

induction

were

labeled

of 5 and,

in ice-water,

breaks

to serve

phages

of phage

by growing

Magnesium

frozen

by thermal

Other

and Abelson

prepared

were

rapidly

( 10 mM ),

) for

from

M EDTA.

at 37'C.

X DNA ( approx.

and reaction

lear

obtained

obtained

10-l' labeling

of infection

paste

( 50 pg ) was added

samples

were

prepared

dithiothreitol

54,000

and 0.002

fractions

double-strand

NaCl,

of Thomas

the flasks

and the cell

added

were

Lambda DNA, uniformly

at a multiplicity

shaking,

media

14

cells

shaking

added

media.

pH 7.5,

Y

Radioactive

by the method

Phage-infected per ml,

x strains

were

in strain

Yloml.

in L.B.

was prepared NaCl,

Other

DNA isolation

state

of strain

lytic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and M. Gold.

High (6)

1,197O

marker in this

of 10% of the

DNA.

Vol.41,No.

substrate

molecules

During

mixtures

were

could

nuclease highly

under

standard

purification,

14 C and 3H-labeled

that

BIOCHEMICAL

1,197O

pooled

for

be analyzed is

purified

conditions.

when larger

DNA's

summarized fraction

were

numbers

alternated

sedimentation per

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

as substrate analysis.

centrifugation

in Table

I.

of protein

of endonuclease

Fraction 1. Alumina extract Streptomycin 3. Ammonium sulfate 4. Alumina Cy 5. DF.AE concentrate 6. Phosphocellulose

manner,

to be assayed,

pairs the number

The purification action

of reaction of fractions

of the endo-

of the enzyme,

the most

was used.

Table Summary of purification

on the

were

and consecutive

In this

was doubled.

In studies

fractions

I from AmlO-infected

Total units (x10-3) 20

Protein (mdml) 30

g. coli

1100

Specific (units/mg

activity protein) 14.8

11

2.

21 19.8

55

19

12.3

concentrate

4

0.15

3

95 200 1025

19800

All procedures were carried out at 0-4'C. Buffer A is 0.05 M h-is pH 7.5, 0.005 M 2-mercaptoethanol, 0.001 M EDTA. Buffer B is 0.005 M Kdphosphate buffer pH 6.5 containing mercaptoethanol and EDTA as in buffer A and 20% (v/v) glycerol. Freshly frozen cells (15 g) were ground with alumina to a smooth paste and the paste was extracted with buffer A. Alumina was removed by centrifugation. The extract (45 ml, OD260: 2501~~) was precipitated by the addition of 0.25 volumes of 5% streptomycin sulfate. The precipitate was discarded after centrifugation and the supernate was fractionated by precipitation with solid ammonium sulfate Virtually all the activity was precipitated between 30 -55% saturation (AS). with AS and the precipitate obtained, collected by brief centrifugation at 100,000 x g, was dissolved in 4 ml of buffer A. The AS fraction was dialyzed extensively against 200 volumes of buffer A. To 4.2 ml of this dialyzate were added 2.1 ml of alumina Cy gel (50 mg solids per ml) and, after 10 min of gentle mixing, the suspension was centrifuged and the solids discarded. The alumina Cy supernate was applied to a column of DF.AE cellulose (2 x 9 cm) equilibrated with buffer A. The column was washed with buffer A and the activity was recovered in the wash. The active fractions were pooled (15 ml total) and concentrated by The dialyzate negative pressure dialysis against buffer B to a volume of 3 ml. was applied to a column of phosphocellulose (1.5 x 10 cm) equilibrated with buffer B. The column was washed with buffer B (100 ml) and buffer B containing, The activity was first, 0.12 M KCl(50 ml) and, then, 0.2 M KC1 (40 ml). recovered in the latter fractions of the 0.2 M effluent. The active fractions were pooled (10 ml total) and concentrated to 1 ml by negative pressure dialysis against buffer A containing 50% (v/v) glycerol. The phosphocellulose concentrate 50% activity after a period of 2 months. was stored at -20°, retaining

65

Vol. 41, No. 1, 1970

Results

BIOCHEMICAL

and Discussion

Biological

source

listed

in Table

of endonuclease. II

resulted

extracts.

Extracts

from

catalyze

a significant

inactive

extracts

not

to detection

lead

5 min after

phage

here.

or cells

fractionation

mechanism

The endonuclease

it

tested

is

not

known whether

If

the endonuclease

be outside ( attachment

site

bacterial

primarily

A simple

interpretation, gene, tested,

( or

are

adjacent

did

be detected

is not

gene for

encoded

at

determined

the enzyme

in the A chromosome

II

).

cistrons

If

could

segments

the

reside

is of

proper,

present

genes

that

in either

the

on the chromosomes

x~I

deficit

mechanism

and highly

site

extremity

results. 66

infection

deleted

Secondly DNA, site

on the

endonuclease in all

the u

DNA segment.

by simultaneous

gene

adjacer

way on the presence

bacterial

of the biotin

by phage

of the

commonly

in trans

structural

deleted.

of bacterial

induction

on the appearance the

First,

of the x attachment

in a positive

site-proximal

are

stretches

the

of h genes

may depend

- that

amplified

attachment

for

of A genes

left

in common.

by specific

) had no effect

at the

properties

and to the right

induction

however

located

replaced

of an attachment

a presumptive

and XL2

the pbio's

could

endonuclease

( Table

of the prophage

on the deficit

the

chromosomes

Amlo

bacterial

);

two general

one possible

Alternatively,

to supplement

our

genes

Thus,

may depend

phage

of purification

in h bio the longest right40, and outside the region deleted in

the active

following

DNA segments

chromosome.

in the

the

and to the right

phage

incorporating

tested.

of the

missing

the activity

bacterial,

proper,

have

proximal

activity

not

e's.

The km's

host

~51 and J.% did

Cy stage

structural

is

to ~111

or in the bacterial

chromosome

the

the X segment

bacterial

the deleted

in crude

Concentration

action

of this

induces

?, genes

with

breaks.

to the alumina

in the expression

origin.

infecting

activity

infected

of double-strand

mutants, since Lb-2 pbio -11 b* specify the endonuclease are

of the

by each of the hw's

infection.

gene must

deletion

of E. coli

of the endonuclease

cells

of activity.

or bacterial

the relevant

uninfected

production

In particular,

phage

Infection

in appearance

and their

The genetic

wise

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of this for

Attemp

infection

activity

with

( Table

the endonuclease

operon, - is not

I

carried established

is a

by all by

II

BIOCHEMICAL

Vol. 41, No. 1,197O

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Endonuclease

Infecting

II

activity

phage

of E, -- coli

Specific

activity ( units/mg

of alumina extracts protein )

1. none

CO.5

2. AC-1

<0.5

3. xb -2

CO.5

4. Xint red ---63 5. h bio +0 6. @b&11


ve27-20

'-

16 15

8. Ab2pbioll 9. mixedly infect AC1 or X2

with

Action

of the

less

5% maximum activity

is

between

6.5 and 7.5

in phosphate

buffer.

High

of DNA breakage,

endonuclease Fig.lA increasing revealed

Manganese

seen with in Tris.

the

latter

Cl-.

concentrations

is

enzyme

not an effective cation.

The reaction

thus

being

distinguished

strictly substitute;

The pH optimum proceeds

of sRNA have no effect

the activity

is

for

equally

well

on either from

that

the rate of

I (8). shows the progress proportion

velocity

sedimenting

a shoulder of intact

of reaction

of h DNA molecules

by sedimentation

a peak appears containing

each of the given strains of h phage prepared and assayed as described in

of DNA by the purified

of magnesium.

is

or extent

with were

Breakage

on the presence

reaction

infected extracts

endonuclease.

dependent than

10-14

Aelo

E_, coli 1100 cells were 20 min, cell and, after Materials and Methods.

fraction

1100

with molecules

0.82

with receive

analysis t 0.01

a relative surviving

times

time.

With

sucrose

as fast

decreases,

time,

one or more double-strand

in neutral

sedimentation

increasing

as intact constant

the

shoulder

breaks

of 0.68

molecules, + 0.01.

becomes

as

Initially,

gradients. marker

an

and

As the

more and more

VoL41,No.

1, 1970

prominent

and,

sedimentation neutral

BIOCHEMICAL

eventually, velocity

gradients

after

DNA is more effective least

two peaks,

sedimentation obtained

the profiles 0.60

when nucleolytic

- 0.70

alkaline

become most

that

of intact

denaturation

in resolving

representing

profiles

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

x DNA fragments,

events

are

of this fully

specifying

Sedimentation

through

?, DNA.

fragments.

of reacted Under

these

readily.

reaction

resemble

patterns

position

(9).

do not

in respect

FRACTIONAL

to

a

substrate

conditions,

can be detected

random

FRACTION NUMBER

in the region

and reneutralization

the product

of the products

dense

at

The

It

SIZE

Fig. 1. (A) Sedimentation profiles of X DNA reacted with 30 units of endonuclease for varying periods of time. Reaction was conducted as described in Materials and Methods and samples were taken for analysis in neutral sucrose gradients at 0 min (w), 10 min (o-----o), and 30 min (-). Sedimentation is from right to left. (B) Molecular size distribution of reaction products in neutral (u) and alkaline (o---o) gradients. Reaction was allowed to continue until approximately 30% of substrate molecules had received at least one double-strand break. Samples of the reaction mix were then analyz'ed by sedimentation in neutral and alkaline gradients. 5 to 20% sucrose alkaline gradients contained 0.1 M NaOH, 0.9 M NaCl, 0.01 M EDTA. Sodium hydroxide ( 0.1 M final concentration ) was added to samples prior to layering on alkaline gradients. Calculations for this type of analysis were according to Meselson and Yuan ( 10 ). The ordinate is arithmetic and units are arbitrary. The abcissa represents the ratio of the molecular sizes of DNA products to that of unreacted marker DNA taken as unity. Molecular sizes were calculated from sedimentation constants using Studier's equations ( 12 ). 68

BIOCHEMICAL

Vol. 41, No. 1, 1970

has been difficult intact

to generate

To compare frequencies Fig.

the number

of fragment

1B represents

fragments

" the reaction

h DNA in "pushing

sizes

in neutral

reaction,

approximately

one half

gradients

have

one or more

Fig.

1B thus

lower

suffered

levels

reveal

weight a moderate

of reaction

over

double-strand

extensive

(all

substrate

mechanism to

of enzyme action

Neither have

duplexes

breaks the role

during of this

been determined.

breaks

in DNA and for

mediate

which functions.

in the endonuclease

described

reason

the

Taken

together,

course

for

this

previously

might

with

intact

a greater

density of

cuts.

However, of the

At this

excess

when reaction type

shown in Fig.lB

are

compatible

with

precede

and are

converted

the genetics

have been

that

catalyze

implicated

@&mutants, both

of its

double-strand

(10,13)

conditionally the

genetic

appear

definition

enzyme.

is pleased to acknowledge the excellent assistance of The research was M. Stewart, and Dr. M. Roy Chowdhury. (MA-3325) from the Medical Research Council of Canada.

9, 734 (1964). Meselson, M., J. Mol. Biol., Signer, E. R., &III. Rev. Microbial., 2& 451 (1968). Manly, K. F., Signer, E. R., and Radding, C. M., Virolow, H., J. Mol. Biol., 2, Durwald, H., and Hoffman-Berling,

69

to

defective

References 1. 2. 3. 4.

a

expression

Acknowledgement The author Mrs. S. Dunbar, Mrs. supported by a grant

is

(10,ll).

studied

provide

of

in neutral

h duplexes),

the results

nor

level

double-strand

plotted.

substrate

The results

incisions

in vivo,

Studies here,

appear

of reaction

roles

of the

At this

of curves

single-strand

biological

30-40%

even more marked. pairs

nuclease

were

of intact

broken),

Endonucleases

degradative

physiological

is

in which

gradients

gradients. over

90% survival

breaks

indistinguishable.

double-strand

of single

than

sucrose

breaks , giving

in alkaline

excess

(more

of single

are virtually

fragments

of

of enzyme. the

that

single-strand

20% that

produced,

breaks.

of the molecules

than

breaks

the case where

one or more double-strand

less

concentrations

and alkaline

for

have received

high

weight

and single-strand

such an analysis

molecular

of molecular with

of double

duplexes

of smaller

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

z 177 (1969). 331 (1968).

and a

Vol. 41, No. 1, 1970

5. 6. 7. 8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gottesman, M. E., and Yarmolinsky, M. B., Cold Spring Harb. Symp. Quant. Biol., 33, 735 (1968). Goldberg, A. R., and Howe, M., Virology, s, 200 (1969). in Nucleic Acid Research" Thomas, C. A., Jr., and Abelson, J., in 'Procedures eds.) p.553, Harper and Row, 1966. (G. L. Cantoni and D. R. Davies, Lehman, I. R., Roussos, G. G., and Pratt, E. A., J. Biol. Chem., 327, 819 (1962). Litwin, S., Shahn, E., and Kozinski, A. W., J. Virology, 5, 24 (1969). Meselson, M., and Yuan, R., Nature, 217, 1110 (1968). C., J. Mol. Biol., ll, 141 (1965). Bernardi, G., and Cordonnier, Studier, F. W., J. Mol. Biol., 11, 373 (1965). Center, M. S., Studier, F. W., and Richardson, C. C., F'roc. Natl. Acad. Sci. U. S., 65, 242 .(1970).

70