Photoreactivating enzyme in the sea urchin egg

Vol.

24,

No.

BIOCHEMICAL

3, 1966

AND

PHOTOREACTIVATING

ENZYME

John Biology

Received

been

1958).

enzyme

organisms

splitting

Stafford, not

1965).

necessarily

imply

may

be used

(Rupert,

1960):

has been for above.

enzyme

that

transforming

DNA,

animal

the

pyrimidine

which

presence

of such

of an extract and

(2) the

either

directly

by a in

,a dimer-

on

DNA

(Jagger

in an organism enzyme.

an enzyme

of an organism destruction

type

However,

involve

acts

review,

in DNA

1966).

not

(for of one

dimers

(Setlow, does

phyla

mechanism

of a photoreactivating

the

the ability

and

biological

of photoreactivation

existence

(1)

Two

that

acts

on

to photoreactivate

of this

photoreoctivating

enzymes. of the

shown the

and

demonstration

the

plant

of light

is indirect

the

is a widespread

evidence

presence

to demonstrate

Photoreactivation

given

Laboratory,

of UV-induced

in the

Thus,

by proteolytic

evidence

EGG*

Setlow

damage

in many

or a light-dependent

UV-inactivated

eggs

splitting

photoreactivation

reaction

ability

K.

COMMUNICATIONS

SEA URCHIN

National Tenn.

(UV)

is considerable

is the

photoreactivating

DNA

THE

Jane

Oak Ridge Oak Ridge,

demonstrated

There

of photoreactivation

criteria

and

of ultraviolet

having

see Jagger,

does

IN

RESEARCH

June 23, 1966

phenomenon,

and

S. Cook

Division,

Photoreactivation

some

BIOPHYSICAL

by

presence

Heretofore,

Blum

UV-induced et al.

delay

(1949)

and

of photoreactivating such

in cleavage by Marshak enzyme

an enzyme

has not

in Arbacia (1949).

in these

been

punctulata

We eggs

demonstrated

have

by the in any

obtained two

criteria

metazoan

tissue.

*

Research sponsored the Union Carbide

by the U. Corporation.

S. Atomic

285

Energy

Commission

under

contract

with

Vol.

24,

No. 3, 1966

BIOCHEMICAL

Preparation cold

of extract.

homogenizing

and

M/15

gentle

were

three

times.

the

then

suspension

last

of 5-10

homogenate

was

retained.

containing

in M/15

experiments.

150 x 2 for

supernatant

contained

contaminating

from

the

Photoreactivation

survival

of transforming

0.25

pg DNA/ml,

was

activity. were

placed

were

added

calf

thymus

DNA

at a concentration

transforming

photoreactivation filtered 7,000 or dark

to eliminate

DNA

to the

from

(at 37”C), all

ergs/mm’/min. incubation,

to the

transform

streptomycin-sensitive

Herriott,

1961).

degradation these

by samples

were

below

samples

ml aliquots

were _.H

3250

were

teflon

final pestle.

supernatant

fluid

supernatant, 1957),

in the

was

pellet

or

improbable

influenzae

in the

exposed

DNA

lamp

that

to about

Various

spot

plate

crude

egg

extract.

assayed

to streptomycin

of the 0.05

to the

For

from

blacklights was

Following for

their

resistance

ml

to protect

intensity

in the dark. and

of

experiments,

incident

a

1 %

dilutions

to illumination The

bearing

at a concentration

In some

was added

A.

The

photoreactivation

either

plates.

incubated

withdrawn

influenzae

spot

nucleases

solution

observed.

ml aliquots.

of 1 mg/ml

wavelengths Control

0.1

in 0.1

the

ml volumes,

porcelain

above

(Layne,

by a germicidal in 0.1

the

it is very

Hemophilus

inactivated

DNA

for

found

effects

DNA.

in white

extract

EDTA was

gauze.

The

assay

homogenization,

Samples

egg

the

before

contribute

marker

1 mM

through

with

and

in

Following

activity.

homogenizer

Biuret

at 4°C.

in the

activity.

activity

wash

out

washed

placed

B-mercaptoethanol,

filtration

15 minutes,

by the

of transforming

resistance

and

no photoreactivating

pH 7, with

last

microorganisms

streptomycin

x 8 for

no photoreactivating

in the supernatant

by

and

5 mM

carried

ovaries

in a glass

at 2,000

buffer,

the

EDTA,

were

COMMUNICATIONS

animals

no photoreactivating

11 mg protein/ml

phosphate

live

1 mM

30 seconds

was homogenized

contained

3 and

Since

from

RESEARCH

from

f ur th er steps

All

at

pellet

removed

containing

separated

was centrifuged The

KCI

7).

were

% eggs

between

diluted

eggs

BIOPHYSICAL

were

M

pH

centrifuged The

The

(0.5

buffer,

agitation,

They

Ovaries

solution

phosphate

AND

ability (Goodgal

about illumination to and

Vol.

24,

No.

3, 1966

Crystalline buffer

prior

bovine

AND

pancreatic

trypsin

BIOPHYSICAL

RESEARCH

(Calbiochem)

COMMUNICATIONS

was dissolved

in M/15

are

in Fig.

phosphate

to use.

Results: apparent

BIOCHEMICAL

The

that

results

there

of a photoreactivation

is a considerable

experiment

increase

in the

number

shown

of transformants

1.

with

It is time

Fig. 1. Number of transformants resulting from irradiated transforming DNA (1% survival) incubated with an Arbacia extract at concentrations of A: 1.1; B: 0.2; and C: 0.1 mg/ml protein.

‘O”WO MINUTES

of exposure of this there

of the

increase

protects

is larger

against

of transformants

of the

shown destroys

same

ratio

greater

(see

has been with

that

in Tables

I and

Table

to the obtained

the Il. (Table

increase

II).

The

ratio

in this

Hence,

dark

laboratory enzyme

in transformation

Incubation I).

addition

of the

extract

either

(1)

287

result

at 37°C the

active

extent

in the thymus

dark

DNA number

is approximately

maximum from

the

maximum

incubation

extracted is the

and

of calf

rate

In addition,

decrease

of the

after

the

extract.

The

the

after

that

incubation,

since

number

a photoreactivating

dark

concentration.

extract,

also

of the

after

extract

in the

and

concentration

of transformants

illumination

Evidence

its activity

with

illumination,

to the

loss of activity

after

DNA

number

to nucleases this

to blacklight

proportional

in the

due

a similar

mixture

is roughly

decrease

is presumably

20;

reaction

is a decrease

of this

INCUBATION

photoreactivation

yeast.

of enzyme for 30 minutes component

activity

is

prior

to assay

is very

heat

Vol.

24,

No. 3, 1966

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE I by heat of photoreactivating capacity of Arbacia for 2. influenzae transforming DNA.

Inactivation

Treatment before

AND

of material assay

Light during

extracts

conditions PR assay

No. of transformants/ml

Extract;

30 minutes

at O’C

L

2167

Extract;

30 minutes

at 0°C

D

448

Extract;

30 minutes

at 37’C

L

891

Extract;

40 minutes

at 60°C

L

946

Extract;

40 minutes

at 60°C

D

863

D

940

Buffer

Diluted

(no extract)

extract

contained

Incubation

90 minutes

in light

TABLE II by trypsin of photoreactivating capacity of Arbacia for 2. influenzae transforming DNA.

Inactivation

Material

1 mg protein/ml.

Light conditions during PR assay

assayed

(L) or dark

extracts

No. of transformants/ml

Extract

L

502

Extract

D

63

Extract

+ trypsin

L

121

Extract

+ trypsin

D

48

D

70

Buffer

+ trypsin

Diluted extract contained Calf thymus DNA added

0.3 before

mg protein/ml. incubation.

Incubation

288

90 minutes.

60 pg/ml

trypsin.

(D).

Vol.

24, No.

labile,

3, 1966

or

BIOCHEMICAL

(2) the

extract.

In either

of its substrate extract

component

case,

the active

(irradiated with

is greatly

reduced

Discussion extract

and

extract

photoreactivates

there

must

yeast

and

2.

which

damage

Arbacia

1966),

and

mechanism. cause

cleavage

heat

lability

and

of the

crude

stabilized

in the

presence

assayed

at 37’C.

If the

its photoreactivating

transforming

the

overlap

in the

likely in Arbacia

1963). same

types

result

that

the

that

most

punctulata

enzyme Since

extent of lesions

or all ore

extract of the pyrimidine

the

as the

ability

of monomerization Arbacia

of the

to an enzyme.

by yeast

of transforming

is the

sensitivity

is due

DNA

to approximately

be inferred

trypsin

capacity

(Setlaw,

extract

It is therefore delay

illumination,

Photoreactivation

by yeast it may

blacklight

A radiation

complete,

extracts. DNA

2537

DNA

if not

is readily

COMMUNICATIONS

components

is presumobly

its activity

influenzae

from this

by other

its photoreactivating

of E.

be a large,

influenzae

(Setlow, some

of the

The

that

RESEARCH

II).

conclusions.

Photoreactivation 90%

during

(Table

indicate

around

component since

trypsin

BIOPHYSICAL

is inactivated

DNA),

is incubated

capacity

Arbacia

active

AND

repairs Arbacia yeast

repaired

enzyme, by the

in UV-irradiated of pyrimidine

probably

acts

dimers by the

photoreoctivable

lesions

dimers.

REFERENCES

Blum,

H. F., G. M. Loos, J. P. Price, and J. C. Robinson, Nature, -164, 1011 (1949). J. Gen. Physiol.,s, 1201 (1961). Goodgaf, 5. H., and R. M. Herriott, Jagger, J., Bacterial. Rev.,g, 99 (1959). Jagger, J., and R. S. Stafford, Biophys. J., 2, 75 (1965). Layne, E., Methods in Enzymology, Vol. III, p. 447, Academic Press, Inc., New 1957. Marshak, A., Biol. Bull., 97, 315 (1949). Rupert, C. S., J. Gen. phpiol., 43,573 (1960). Setlow, J. K., Photochem. Photobzl., 2, 393 (1963). Setlow, J. K., Current Topics in Radiatzn Research, Vol. II. p. 195, North-Holland Publishing Co., Amsterdam, 1966.

289

York,